Substituted Cyclopenta Pyrimidine Bicyclic Compounds Having Antitmitotic And/Or Antitumor Activity And Methods Of Use Thereof

ABSTRACT

The present invention provides substituted cyclopenta and cyclopentyl pyrimidine bicyclic compounds of Formula III, 
     
       
         
         
             
             
         
       
     
     and 5,6-saturated and unsaturated
 
and pharmaceutically acceptable salts, prodrugs, solvates, and hydrates thereof, having antimitotic activity, anti-multidrug resistance activity, such as for example P-glycoprotein inhibition, and antitumor activity, and which inhibit paclitaxel sensitive and resistant tumor cells. Also provided are methods of utilizing these compounds for treating tumor cells and inhibiting mitosis of cancerous cells.

CROSS-REFERENCE TO RELATED APPLICATIONS

This continuation-in-part patent application claims the benefit ofpending U.S. patent application Ser. No. 12/170,571, filed on Jul. 10,2008. The entire contents of U.S. patent application Ser. No. 12/170,571is incorporated by reference into this continuation-in-part utilitypatent application as if fully written herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant No. R01CA142868 awarded by the National Institute of Health, National CancerInstitute. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to bicyclic heteroaromatic compounds andtheir methods of use and, more particularly, to bicyclic heteroaromaticcompounds that are antitumor agents that inhibit the function ofmicrotubules (antimitotic agents or mitotic inhibitors) and that haveantitumor activity. These bicyclic heteroaromatic compounds inhibitP-glycoprotein (Pgp) infected tumor cells, and inhibit paclitaxelsensitive and resistant tumor cells. The compounds may be made into acidsalts that are water soluble for providing orally active antitumoragents.

BACKGROUND OF THE INVENTION

Mitosis is the process of nuclear division in eukaryotic cells thatproduces two daughter cells from one parent cell. The daughter cells andthe original parent cell have identical chromosomes and DNA. Generally,cancer is a disease of mitosis. It is believed that cancer begins when asingle cell is converted from a normal cell to a cancer cell. this isoften due to a change in function of one or more genes that normallyfunction to control cell growth. The cancer cells proliferate byrepeated, and uncontrolled mitosis, in contrast to normal cells whichundergo only about 20 to 50 generations of replication and then cease. Atumor may be thought of a mass of unhealthy cells that are dividing andgrowing in an uncontrolled way.

Microtubules are long, protein polymers that are hollow, tube-likefilaments found in certain cell components such as the mitotic spindle.Each microtubule is composed of repeating subunits of the proteintubulin. Microtubules aggregate to form spindle fibers. During mitosis,cells use their spindle fibers to line up chromosomes, make copies ofthem, and divide into new cells with each new daughter cells having asingle set of chromosomes. The polymerization dynamics of microtubulesplay a pivotal role in this process as part of cell replication. Thecrucial involvement of microtubules in mitosis makes them a target forantitumor agents. Antitumor agents that inhibit the function ofmicrotubules are known as antimitotic agents.

Many classes of antimitotic agents are known. One such class is thevinca alkaloids exemplified by vincristine, vinblastine, vindesine, andvinorelbine. The vinca alkaloids are used in the treatment of leukemias,lymphomas, and small cell lung cancer. Another class of antimitoticagents are the taxanes, exemplified by paclitaxel (commerciallyavailable from Bristol-Myers Squibb Company under the tradename TAXOL®)and docetaxel. The taxanes are useful in the treatment of breast, lung,ovarian, head and neck, and bladder carcinomas. Colchicine typifiesanother class of antimitotic agents. Colchicine, while not used as anantitumor agent, is a microtubule polymerization inhibitor. Lastly, thecombrestatins are another class of antitumor agents. Antimitotic agentssuch as the vinca alakaloids, colchicine, colcemid, and nocadazol blockmitosis by keeping the mitotic spindle from being formed. These agentsbind to the tubulin and inhibit polymerization, preventing cells frommaking the spindles they need to move chromosomes around as they divide.In contrast, paclitaxel binds to the tubulin protein of microtubules,locking the microtubules in place and inhibiting their depolymerization.With the mitotic spindle still in place, a cell may not divide intodaughter cells.

Multidrug or multiple drug resistance (MDR) is a major drawback ofcancer chemotherapy. Ultimate failure of chemotherapy often times occurswith the use of antimitotic agents due to MDR. MDR may be inherentlyexpressed by some tumor types while others acquire MDR after exposure tochemotherapy. P-glycoprotein (Pgp) is a 170 kilodalton (kDa) proteinthat belongs to the ATP-binding cassette superfamily of transporters.Pgp has been implicated as a primary cause of MDR in tumors. Pgps areefflux transporters found in the gut, gonads, kidneys, biliary system,brain, and other organs. A series of homologous proteins termedmultidrug-resistance proteins (MRPs) are also known. MRPs are associatedwith MDR in tumors. The first MRP termed MRP1 was identified in a drugresistant lung cancer cell line that expressed Pgp. All of thesetransporters bind drugs within cells and release them to theextracellular space using ATP. Tumor cells pre-exposed to cytotoxiccompounds often allow the cells to manifest resistance in the presenceof the cytotoxic drug. Overexpression of Pgp has been reported in anumber of tumor types, particularly after the patient has receivedchemotherapy, indicating the clinical importance of Pgp in MDR. Theclinical significance of Pgp along with its limited expression in normaltissues makes Pgp a viable target for inhibition to reverse MDR.

While antimitotic agents have shown to be some of the most successfulagents against malignancies, resistance, both intrinsic and acquired,often results in treatment failures. Thus, there exists a need todevelop new compounds that possess antimitotic activity, anti-multidrugresistance activity, and antitumor activity, that may be used alone as asingle agent in the treatment of cancer, or in combination withchemotherapeutic agents, including antimitotic agents, that shallinhibit mitosis in a wide variety of cells, including cells that aresubject to MDR. There is a need, therefore, for single compounds whichprovide the desired antimitotic, anti-multidrug resistance and antitumoractivities with a high degree of selectivity and low toxicity, and thatare effective inhibitors of paclitaxel sensitive and resistant tumorcells.

SUMMARY OF THE INVENTION

The present invention meets the above need by providing bicycliccompounds having antimitotic activity, anti-multidrug resistanceactivity (for example, Pgp inhibition), and antitumor activity in asingle molecule so that significant drawbacks of different aspects ofdrug transport of two or more drugs to their targets, additive orsynergistic toxicities of two or more different drugs, resistance ofcancer cells to a particular drug, as well as the cost associated withtwo or more drugs, is circumvented.

The present invention provides single compounds that exhibit antimitoticactivity, anti-multidrug resistance activity (for example, Pgpinhibition), and antitumor activity in tumor cells, such as, withoutlimitation, leukemia, non-small cell lung cancer, colon cancer, centralnervous system cancer, melanoma, ovarian cancer, renal cancer, prostatecancer and breast cancer; and other proliferative diseases anddisorders.

The present provides single compounds having a combinatorialchemotherapeutic potential of both antimitotic activity, anti-multidrugresistance activity, and antitumor activity, and which inhibitpaclitaxel sensitive and resistant tumor cells.

In an aspect of the present invention, there is provided a compound ofFormula III:

and 5,6-saturated and unsaturatedwherein the five membered ring may be saturated or unsaturated withrespect to bond 5-6;

R₁ and R₂ may be the same or different and comprises one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

R₅ comprises one of R₁;

R₆ comprises one of R₁;

X comprises one of (a) NR₄, (b) an oxygen (O), and (c) a R₇CR₄; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent. Preferably, the compound ofFormula III as described herein, comprises pharmaceutically acceptablesalts, prodrugs, solvates, or hydrates thereof.

In another embodiment of the present invention, the compound of FormulaIII, as described herein, is provided, wherein R₅, R₆, and R₃ are thesame moiety. Preferably, a further embodiment comprises wherein thecompounds are pharmaceutically acceptable salts, prodrugs, solvates, orhydrates thereof.

In another embodiment of the present invention, the compound of FormulaIII, as described herein, is provided, wherein R₇, R₄, R₆, and R₅ areeach a moiety comprising one of R₁. Preferably, a further embodimentcomprises wherein the compounds are pharmaceutically acceptable salts,prodrugs, solvates, or hydrates thereof.

In another aspect of the present invention, there is provided a compoundof Formula IV:

-   -   and 6,7-saturated and unsaturated        wherein the five membered ring may be saturated or unsaturated        with respect to bond 6-7;

R₁ and R₂ may be the same or different and comprises one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

R₅ comprises one of R₁;

R₆ comprises one of R₁;

X comprises one of (a) a NR₄, (b) an oxygen (O), and (c) R₇CR₄; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e is zero). Preferably, thecompound of Formula IV, as described herein, comprises apharmaceutically acceptable salt, prodrug, solvate, or hydrate thereof.

In another embodiment of this invention, the compound having Formula IV,as described herein, further comprises wherein R₅, R₆, and R₃ are thesame moiety. Preferably, in another embodiment, the Formula IV whereinR₅, R₆, and R₃ are the same moiety, comprises a pharmaceuticallyacceptable salt, prodrug, solvate, or hydrate thereof.

In another embodiment of this invention, the compound of Formula IV, asdescribed herein, is provided comprising wherein R₆ and R₅ are the samemoiety and comprise one of R₁. Preferably, in another embodiment of thisinvention, the compound of the Formula IV wherein R₆ and R₅ are the samemoiety, comprises a pharmaceutically acceptable salt, prodrug, solvate,or hydrate thereof.

In another embodiment of this invention, a method of treating a patienthaving cancer is provided comprising administering to the patient atherapeutically effective amount of a compound of Formula III, asdescribed herein, or a pharmaceutically acceptable salt, prodrug,solvate, or hydrate thereof.

In another embodiment of this invention, a method of treating a patienthaving cancer is provided comprising administering to the patient atherapeutically effective amount of a compound of Formula IV, asdescribed herein, or a pharmaceutically acceptable salt, prodrug,solvate, or hydrate thereof.

In yet another embodiment of this invention, a method for inhibiting themitosis of one or more cancerous cells is provided comprising subjectingone or more live cancerous cell to a mitotic inhibitory amount of acompound of Formula III, as described herein, or a pharmaceuticallyacceptable salt, prodrug, solvate, or hydrate of the compound of FormulaIIII, for effecting the inhibition of mitosis of the cancerous cell(s).

Another embodiment of this invention provides a method for inhibitingthe mitosis of one or more cancerous cells comprising subjecting atleast one live cancerous cell to a mitotic inhibitory amount of acompound of Formula IV, as described herein, or a pharmaceuticallyacceptable salt, prodrug, solvate, or hydrate of a compound of FormulaIV, for effecting the inhibition of mitosis of the cancerous cell(s).

In a more preferred embodiment of this invention, a compound of FormulaIII:

and 5,6-saturated and unsaturated

with respect to the five membered ring;

R₁ and R₂ may be the same or different and each is one of (a) a hydrogen(H), (b) an alkyl having from one to ten carbon atoms and having astraight or branched configuration, and wherein the alkyl is partiallyor completely saturated, or a substituted alkyl having from one to tencarbon atoms, (c) a cycloalkyl having from three to ten carbon atoms, ora substituted cycloalkyl having from three to ten carbon atoms, (d) analkylcycloalkyl, or a substituted alkylcycloalkyl, (e) an aryl, or asubstituted aryl, (f) an alkylaryl, or a substituted alkylaryl (g) aheteroaryl, or a substituted heteroaryl, (h) an alkylheteroaryl, or asubstituted alkylheteroaryl, (i) an aromatic, or a substituted aromatic,and (j) a heteroaromatic, or a substituted heteroaromatic, and whereineach substituent of any said substituted group is the same or differentand is selected from the group consisting of a straight or branchedalkyl, alkenyl, or alkynyl, a cyclic or alicyclic group having from oneto six carbon atoms, a heterocyclic group having from one to six carbonatoms, an alkoxy group, an aryloxy group, an alkyloxyaryloxy group, anaryl group, an amine, a halogen, a phenol, a naphthalene, a piperidine,a pyrrole, a ketone, a methylalkyl ketone, and a trifluoromethyl ketone,and wherein each of said substituents may itself be substituted, andwherein any of said substituents may be optionally attached by a CH₂bridge, and wherein the substituent may be optionally partially orcompletely saturated or unsaturated when it is not represented by saidhalogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

wherein R₁ and R₂ are not each hydrogen (H) when Y is N;

R₃ is one of (a) a hydrogen (H), (b) a halogen, (c) an alkyl having fromone to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R is one of R₁, and wherein R₇ andR₈ may be the same or different and is one of R₁;

R₅ is one of R₁, except when R₆ is a hydrogen (H), heteroaryl, orphenyl, wherein the phenyl and heteroaryl are optionally substitutedwith 1-2 moieties independently selected from the group consisting ofhalogen, (C₁-C₄)alkyl, (C₁-C₄)fluoroalkyl, (C₁-C₄)alkoxy,(C₁-C₄)alkylamine, and (C₁-C₄)dialkylamine, then R₅ is not (i) ahydrogen (H), or (ii) a phenyl group substituted at any position with asubstituent selected from the group consisting of (a) a hydrogen, (b) ahalogen, (c) CN, (d) OH, (e) NH₂, (f) (C₁-C₄)alkyl, (g)(C₃-C₆)cycloalkyl, (h) (C₁-C₄)fluoroalkyl, (i) (C₁-C₄)alkoxy, (j)(C₁-C₄)alkylamine, (k) (C₁-C₄)dialkylamine, (l) C(O)OH, (m) C(O)—NH₂,(n) C(O)—(C₁-C₄)alkyl, (O)C(O)—(C₁-C₄)fluoroalkyl, (p)C(O)—(C₁-C₄)alkylamine, and (q) C(O)—(C₁-C₄)alkoxy;

R₆ is one of R₁;

X is a carbon (C) or a CR₄; and

Y is one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur (S), and(d) a CR₆, wherein R₆ is one of R₁ and R₃ except when Y is CR₆ then saidR₆ is not H when R₁ and R₂ are each H, and wherein when Y is O or S thenR₂ is absent or is zero. Further, this invention provides for thecompound of Formula III comprising pharmaceutically acceptable salts,hydrates, and solvates thereof. More preferably, this invention providesfor a compound of Formula III, as described herein, wherein R₁ is eithera hydrogen or said alkyl having from one to ten carbon atoms and R₂ is asubstituted naphthyl, or wherein R₁ is a substituted naphthyl and R₂ iseither a hydrogen or said alkyl having from one to ten carbon atoms.Most preferably, this invention provides the compound of Formula IIIwherein the substituted naphthyl is a methoxynaphthyl group and whereinthe 5,6 bond is a single bond.

Another embodiment of this invention provides a compound comprisingFormula IV:

-   -   and 6,7-saturated and unsaturated

wherein R₁ and R₂ may be the same or different and comprises one of (a)a hydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

R₅ comprises one of R₁;

R₆ comprises one of R₁;

X is a carbon (C) or a CR₄; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃. Further, thisinvention provides for a compound of Formula IV comprisingpharmaceutically acceptable salts, solvates, and hydrates thereof. Morepreferably, this invention provides for the compound of Formula IV, asdescribed herein, wherein R₁ is either a hydrogen or said alkyl havingfrom one to ten carbon atoms and R₂ is a substituted naphthyl, orwherein R₁ is a substituted naphthyl and R₂ is either a hydrogen or saidalkyl having from one to ten carbon atoms. Most preferably, thisinvention provides for a compound of Formula IV, as described herein,wherein said substituted naphthyl is a methoxynaphthyl.

BRIEF DESCRIPTION OF THE DRAWINGS

A full understanding of the invention can be gained from the followingdescription of the preferred embodiments when read in conjunction withthe accompanying drawings in which:

FIGS. 1 a, 1 b, and 1 c show the effects of various compounds of thepresent invention on a cancer cell line's cell cycle distribution.

FIG. 2 shows a microtubule depolymerization immunofluorescence assay ofA10 rat smooth muscle tumor cell line following treatment with compoundsof the present invention, namely, Sample IDs AAG1, AAG7, and AAG16.

FIG. 3 shows the chemical structures of six compounds of the presentinvention, namely, Sample IDs AAG1, AAG7, AAG12, AAG16, AAG20, andAAG26.

FIG. 4 shows the biological effects of the compounds of the presentinvention.

FIGS. 5 a and 5 b shows the results of the National Cancer Institute's55 preclinical in vitro tumor screening panel evaluating a compound ofthe present invention, namely, Sample ID AAG 1.

FIGS. 6 a, 6 b and 6 c show individual dose response curves ofpercentage growth for each of the cancer cell lines set forth in FIG. 5.

FIG. 7 shows a dose response curve of percentage growth for all of thecell lines shown in FIG. 5.

FIG. 8 shows mean graphs for each of the cancer types and correspondingcell lines shown in FIG. 5.

FIG. 9 shows structures of know microtubule targeting agents.

FIG. 10 shows the results and interpretation of a NOESY study of a morepreferred methoxynaphthyl substituted cyclopenta pyrimidine compound 11of the present invention.

FIG. 11 shows the results and interpretation of a NOESY study of a morepreferred methoxynaphthyl substituted cyclopenta pyrimidine compound 13of the present invention.

FIG. 12 shows the results and interpretation of a NOESY study of a morepreferred methoxynaphthyl substituted cyclopenta pyrimidine compound 14of the present invention.

FIGS. 13 a and b show MOE (2012) simulation of the low energy conformerof the more preferred methoxynaphtyl substituted cyclopenta pyrimidinecompounds 11 and 13 of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides bicyclic compounds having antimitoticactivity, anti-multidrug resistance activity (for example, Pgpinhibition), and antitumor activity, and which inhibit paclitaxelsensitive and resistant tumor cells in a single molecule and methods ofuse thereof.

The present invention provides a compound of the Formula III:

and 5,6-saturated and unsaturatedwherein the five membered ring may be saturated or unsaturated withrespect to bond 5-6;

R₁ and R₂ may be the same or different and comprises one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

R₅ comprises one of R₁;

R₆ comprises one of R₁;

X comprises one of (a) NR₄, (b) an oxygen (O), and (c) R₇CR₄; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero).

In another embodiment of this invention the compound having Formula III,as described herein, further comprises wherein R₅, R₆, and R₃ are thesame moiety.

In yet another embodiment of this invention the compound of Formula III,as described herein, further comprises wherein R₇, R₄, R₆, and R₅ areeach a moiety comprising R₁.

Preferably, the compounds of Formula III, as described herein, arepharmaceutically acceptable salts, prodrugs, solvates, or hydratesthereof.

Another embodiment of this invention provides a compound of Formula IV:

-   -   and 6,7-saturated and unsaturated

wherein the five membered ring may be saturated or unsaturated withrespect to bond 6-7;

R₁ and R₂ may be the same or different and comprises one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

R₅ comprises one of R₁;

R₆ comprises one of R₁;

X comprises one of (a) NR₄, (b) an oxygen (O), and (c) R₇CR₄; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero).

In another embodiment of this invention the compound having Formula IV,as described herein, is provided comprising wherein R5, R6, and R3 arethe same moiety.

In yet another embodiment of this invention the compound having FormulaIV, as described herein, is provided comprising wherein R₆, and R₅ arethe same moiety and comprise one of R₁.

Other embodiments of the present invention provide pharmaceuticallyacceptable salts, prodrugs, solvates, and hydrates of the compounds ofFormulae III and IV. Preferably, the compounds of the present inventionrepresented by Formulae III and IV may be made into acid salts that arewater soluble. Most preferably, these water soluble salts of FormulaeIII and IV may be formulated into an oral dosage forms providing orallyadministered active antitumor agents. In the past, antimitotic agentshave been plagued with water solubility problems, such as for examplebut not limited to Taxol® and combrestastatin, and a variety ofsolubilizing agents have been employed to improve their watersolubility. The present salts of Formulae III and IV overcome such watersolubility problems and are generally completely water soluble.

In another embodiment of this invention, a method of treating a patienthaving cancer is provided comprising administering to the patient atherapeutically effective amount of a compound of Formula III, asdescribed herein, or a pharmaceutical acceptable salt, prodrug, solvate,or hydrate of the compound of Formula III:

and 5,6-saturated and unsaturated

wherein the five membered ring may be saturated or unsaturated withrespect to bond 5-6;

R₁ and R₂ may be the same or different and comprises one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

R₅ comprises one of Ri;

R₆ comprises one of R₁;

X comprises one of (a) NR₄, (b) an oxygen (O), and (c) R₇CR₄; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero). In anotherembodiment of this invention, this method comprises providing thecompound having Formula III, as described herein, wherein R₅, R₆, and R₃are the same moiety, or wherein the compound of Formula III, asdescribed herein, comprises wherein R₇, R₄, R₆, and R₅ are each a moietycomprising R₁.

As used herein, the term “patient” means members of the animal kingdom,including, but not limited to, human beings. As used herein, the term“having cancer” means that the patient has been diagnosed with cancer.

As used herein, the term “therapeutically effective amount” refers tothat amount of any of the present compounds required to bring about adesired effect in a patient. The desired effect will vary depending onthe illness being treated. For example, the desired effect may bereducing tumor size, destroying cancerous cells, and/or preventingmetastasis, any one of which may be the desired therapeutic response. Onits most basic level, a therapeutically effective amount is that amountneeded to inhibit the mitosis of a cancerous cell or to facilitate thereversal of multidrug resistance, particularly, for example due toP-glycoprotein, (ie. an effective mitotic inhibitory amount). Any amountof mitotic inhibition or reversal of multidrug resistance will yield abenefit to a patient and is therefore within the scope of the invention.

In another embodiment of this invention, a method of treating a patienthaving cancer is provided comprising administering to the patient atherapeutically effective amount of a compound of Formula IV, or apharmaceutical acceptable salt, prodrug, solvate, or hydrate of thecompound of Formula IV:

-   -   and 6,7-saturated and unsaturated

wherein the five membered ring may be saturated or unsaturated withrespect to bond 6-7;

R₁ and R₂ may be the same or different and comprises one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

R₅ comprises one of R₁;

R₆ comprises one of R₁;

X comprises one of (a) NR₄, (b) an oxygen (O), and (c) R₇CR₄; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero). Otherembodiments of the present invention provide for this method includingproviding the compound having Formula IV, as described herein,comprising wherein R₅, R₆, and R₃ are the same moiety, or wherein R₆,and R₅ are the same moiety and comprise one of R₁.

Compounds of the present invention covered under Formula III and IV mayalso be administered with one or more additional treatment agents, i.e.,a chemotherapeutic agent. Suitable candidates for the additionalchemotherapeutic agent include for example but are not limited to,paclitaxel, docetaxel, vinca alkaloids, colchicines, colcemid,cisplatin, and nocadazol. The presence of the compound of the presentinvention shall enhance the effectiveness of the chemotherapeutic agentby facilitating the reversal of multidrug resistance, particularly dueto Pgp, and at least partially restoring the sensitivity of tumors toantimitotic agents.

In yet another embodiment of this invention, a method for inhibiting themitosis of one or more cancerous cells is provided comprising subjectingone or more live cancerous cells to an effective inhibitory amount of acompound of Formula III, or a salt, prodrug, solvate, or hydrate of acompound of Formula III:

and 5,6-saturated and unsaturated

wherein the five membered ring may be saturated or unsaturated withrespect to bond 5-6;

R₁ and R₂ may be the same or different and comprises one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

R₅ comprises one of Ri;

R₆ comprises one of R₁;

X comprises one of (a) NR₄, (b) an oxygen (O), and (c) R₇CR₄; and Ycomprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e. is zero), for effectingthe inhibition of mitosis of the cancerous cells.

Another embodiment of this invention provides a method for inhibitingthe mitosis of one or more cancerous cells comprising subjecting livecancerous cells to an effective mitotic inhibitory amount of a compoundof Formula IV, or a salt, prodrug, solvate or hydrate of a compound ofFormula IV:

-   -   and 6,7-saturated and unsaturated

wherein the five membered ring may be saturated or unsaturated withrespect to bond 6-7;

R₁ and R₂ may be the same or different and comprises one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

R₅ comprises one of R₁;

R₆ comprises one of R₁;

X comprises one of (a) a NR₄, (b) an oxygen (O), and (c) R₇CR₄; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃, and whereinwhen Y comprises O or S then R₂ is absent (i.e is zero), for effectingthe inhibition of mitosis of the cancerous cells.

As used herein, the term “lower alkyl” group refers to those lower alkylgroups having one to about ten carbon atoms, such as for example methyl,ethyl, propyl, butyl, pentyl, hexyl, cyclopropyl, cyclobutyl,cyclohexyl, cyclopropylmethyl or cyclobutylmethyl groups. Alkyl groupssharing one to about six carbon atoms are preferred. These lower alkylgroups are straight chain, branched chain or cyclic (alicyclichydrocarbon) arrangements. The carbon atoms of these straight chain,branched chain or cyclic arranged alkyl groups may have one or moresubstituents for the hydrogens attached to the carbon atoms.

As used herein, the term “heteroalkyl” refers to alkyl chains from oneto about 3 atoms where one or more of the carbons has been replaced withnitrogen, oxygen or sulfur, Thus “heteroalkyl” groups will include, forexample, C—C—N, C—S, S—C, C—O, C—C—O, O—C, N—C—C, N—C^(═)C and othervarious combinations, as will be apparent to one skilled in the art. Theabove list is not meant to be exhaustive, and many combinations arecontemplated as within the scope of the present invention.

The term “aryl” groups, as used herein, refers to compounds whosemolecules have an aromatic ring structure, such as the six-carbon ringof benzene, or multiple rings which are either fused or unfused, such ascondensed six-carbon rings of other aromatic derivatives. The term“aryl” is also defined to include diaryl, triaryl and polyaryl groups,which would have two, three or more rings, respectively. Thus, suitablearyl groups would include, for example, phenyl, biphenyl, naphthyl,phenanthrene, anthracene groups and aryl oxyaryl groups. This list isnot meant to be exhaustive, and any aryl group, as these terms aredefined above and commonly understood in the art, are within the scopeof the present invention.

The term “heteroaryl” refers to aromatic ring structures having at leastone atom in the ring which is not carbon, such as oxygen, nitrogen orsulfur. “Heteroaryls” as used herein also refers to aromatic ringstructures that are part of larger ring structures, such as two or threemember ring systems, which may be fused or unfused, in which one of therings is as described above. Thus, “heteroaryl” refers to ring systemsin which one or more rings contain a heteroatom and one or more rings donot. It will be understood that this list is not meant to be exhaustive,and that any heteroaryl group, as these terms are defined above andcommonly understood in the art, are within the scope of the presentinvention. The heteroaryl ring systems may be fused ring systems orunfused. Examples of heteroaryl ring systems include, for example butare are not limited to, pyridine, quinoline, isoquinoloine, pyrrole,thiophenes, furans, imidazoles, and the like, as well as fused ringstructures having rings of different sizes, such as benzofurans,indoles, purines, and the like.

Also included within the scope of the present invention are alicyclicgroups, as that term is understood in the art, and heterocyclic groups.As used herein, the term “heterocyclic group” refers to non-aromaticcyclic substituents in which one or more members of the ring is notcarbon, for example oxygen, sulfur or nitrogen.

The terms “alkylaryl” (or “alkaryl”) or “alkylheteroaryl” as used hereinrefer to groups having an alkyl moiety attached to an aryl or heteroarylring. The alkyl moiety is preferably a straight, branched or cyclicalkyl group having one to about six carbon atoms. This alkyl moiety mayalso contain oxygen, nitrogen or sulfur, and therefore may be an alkoxygroup. The aryl or heteroaryl moiety of the alkylaryl group is asubstituted or unsubstituted aryl or heteroaryl group, as these termsare described above. As used herein, the terms “alkylaryl” or“alkylheteroaryl” will also be used to refer to arylalkyl groups orheteroarylalkyl groups, as those terms are understood in the art, anddenotes attachment of such a substituent at either the alkyl or the arylportion of the group. Thus, for example, a benzyl group would beembraced by the term “alkylaryl”.

Any of the cyclic substituents described above, such as the aryl,heteroaryl, alkylaryl, alkylheteroaryl, alicyclic, or heterocyclicgroups are optionally substituted with one or more substituents aslisted above. In the case of more than one substituent, the substituentsare independently selected. “Alkoxy groups” and “alkyl groups” includestraight or branched chains having up to about ten members. “Halogen”refers to chlorine, bromine, iodine and fluorine. “Aryl and heteroarylgroups” are as described above. When a carboxylic acid is a substituent,it will be appreciated that the moiety represents an acid such asbenzoic acid.

As used herein, the terms “aroyl” or “heteroaroyl”, such as when usedwithin the term p-aroyl-L-glutamate, refers to benzoyl, napthoyl,thiophenoyl, furophenoyl, pyrroyl, and any other “aroyl” or“heteroaroyl” as these terms would be understood by one skilled in theart. “Aroyl” and “heteroaroyl” are generally defined in the art as anaromatic or heteroaromatic compound having a carbonyl moiety. As usedherein, the term “glutamate” will be understood as representing both theester form (glutamate) and the acid form (glutamic acid).

It will appreciated by those skilled in the art that a general formuladepicting compounds having side chains with adjacent carbons having adouble bond will result in both cis and trans isomers as possiblestructures. Both the cis and trans isomers, and mixtures thereof, of anysuch compound within the broad general formula described in Formulas IIIand IV are contemplated as being within the scope of the presentinvention.

A preferred form of Formula IV is shown in FIG. 3, Sample ID AAG12.

In a more preferred embodiment of this invention, a compound of FormulaIII:

and 5,6-saturated and unsaturated

with respect to the five membered ring;

R₁ and R₂ may be the same or different and each is one of (a) a hydrogen(H), (b) an alkyl having from one to ten carbon atoms and having astraight or branched configuration, and wherein the alkyl is partiallyor completely saturated, or a substituted alkyl having from one to tencarbon atoms, (c) a cycloalkyl having from three to ten carbon atoms, ora substituted cycloalkyl having from three to ten carbon atoms, (d) analkylcycloalkyl, or a substituted alkylcycloalkyl, (e) an aryl, or asubstituted aryl, (f) an alkylaryl, or a substituted alkylaryl (g) aheteroaryl, or a substituted heteroaryl, (h) an alkylheteroaryl, or asubstituted alkylheteroaryl, (i) an aromatic, or a substituted aromatic,and (j) a heteroaromatic, or a substituted heteroaromatic, and whereineach substituent of any said substituted group is the same or differentand is selected from the group consisting of a straight or branchedalkyl, alkenyl, or alkynyl, a cyclic or alicyclic group having from oneto six carbon atoms, a heterocyclic group having from one to six carbonatoms, an alkoxy group, an aryloxy group, an alkyloxyaryloxy group, anaryl group, an amine, a halogen, a phenol, a naphthalene, a piperidine,a pyrrole, a ketone, a methylalkyl ketone, and a trifluoromethyl ketone,and wherein each of said substituents may itself be substituted, andwherein any of said substituents may be optionally attached by a CH₂bridge, and wherein the substituent may be optionally partially orcompletely saturated or unsaturated when it is not represented by saidhalogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

wherein R₁ and R₂ are not each hydrogen (H) when Y is N;

R₃ is one of (a) a hydrogen (H), (b) a halogen, (c) an alkyl having fromone to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R is one of R₁, and wherein R₇ andR₈ may be the same or different and is one of R₁;

R₅ is one of R₁, except when R₆ is a hydrogen (H), heteroaryl, orphenyl, wherein the phenyl and heteroaryl are optionally substitutedwith 1-2 moieties independently selected from the group consisting ofhalogen, (C₁-C₄)alkyl, (C₁-C₄)fluoroalkyl, (C₁-C₄)alkoxy,(C₁-C₄)alkylamine, and (C₁-C₄)dialkylamine, then R₅ is not (i) ahydrogen (H), or (ii) a phenyl group substituted at any position with asubstituent selected from the group consisting of (a) a hydrogen, (b) ahalogen, (c) CN, (d) OH, (e) NH₂, (f) (C₁-C₄)alkyl, (g)(C₃-C₆)cycloalkyl, (h) (C₁-C₄)fluoroalkyl, (i) (C₁-C₄)alkoxy, (j)(C₁-C₄)alkylamine, (k) (C₁-C₄)dialkylamine, (l) C(O)OH, (m) C(O)—NH₂,(n) C(O)—(C₁-C₄)alkyl, (O)C(O)—(C₁-C₄)fluoroalkyl, (p)C(O)—(C₁-C₄)alkylamine, and (q) C(O)—(C₁-C₄)alkoxy;

R₆ is one of R₁;

X is a carbon (C) or a CR₄; and

Y is one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur (S), and(d) a CR₆, wherein

R₆ is one of R₁ and R₃ except when Y is CR₆ then said R₆ is not H whenR₁ and R₂ are each H, and wherein when Y is O or S then R₂ is absent oris zero. Further, this invention provides for the compound of FormulaIII comprising pharmaceutically acceptable salts, hydrates, and solvatesthereof. More preferably, this invention provides for a compound ofFormula III, as described herein, wherein R₁ is either a hydrogen orsaid alkyl having from one to ten carbon atoms and R₂ is a substitutednaphthyl, or wherein R₁ is a substituted naphthyl and R₂ is either ahydrogen or said alkyl having from one to ten carbon atoms. Mostpreferably, this invention provides the compound of Formula III whereinthe substituted naphthyl is a methoxynaphthyl group and wherein the 5,6bond is a single bond.

Another embodiment of this invention provides a compound comprisingFormula IV:

-   -   and 6,7-saturated and unsaturated

wherein R₁ and R₂ may be the same or different and comprises one of (a)a hydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen;

wherein (i) and (j) may optionally be attached to Y via a CH₂ bridge;

R₃ comprises one of (a) a hydrogen (H), (b) a halogen, (c) an alkylhaving from one to ten carbon atoms and having a straight or a branchedconfiguration, and wherein the alkyl is partially or completelysaturated; (d) an NH₂, (e) an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR,(i) an SH, and (j) an SR, and wherein R comprises one of R₁, and whereinR₇ and R₈ may be the same or different and comprise one of R₁;

R₄ comprises one of (a) R₁, (b) a halogen, (c) a mono-, di-, tri- ortetra-substituted alkyl, and (d) an alkyloxy, and wherein R₁ is H or alower alkyl and R₂ is H or a lower alkyl then R₄ comprises one of (a) aNR₆R₇, (b) a SR₆, (c) a OR₆, and (d) a CHR₆R₇, wherein R₆ and R₇ may bethe same or different and comprise one of R₁ and R₂;

R₅ comprises one of R₁;

R₆ comprises one of R₁;

X is a carbon (C) or a CR₄; and

Y comprises one of (a) a nitrogen (N), (b) an oxygen (O), (c) a sulfur(S), and (d) a CR₆, wherein R₆ comprises one of R₁ and R₃. Further, thisinvention provides for a compound of Formula IV comprisingpharmaceutically acceptable salts, solvates, and hydrates thereof. Morepreferably, this invention provides for the compound of Formula IV, asdescribed herein,

wherein R₁ is either a hydrogen or said alkyl having from one to tencarbon atoms and R₂ is a substituted naphthyl, or wherein R₁ is asubstituted naphthyl and R₂ is either a hydrogen or said alkyl havingfrom one to ten carbon atoms. Most preferably, this invention providesfor a compound of Formula IV, as described herein, wherein saidsubstituted naphthyl is a methoxynaphthyl.

Proliferative diseases and/or disorders that may be treated according tothe methods of the present invention include, without limitation,leukemia, non-small cell lung cancer, colon cancer, central nervoussystem (CNS) cancer, melanoma, ovarian cancer, renal cancer, prostatecancer, and breast cancer.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the patients being treated, each unitcontaining a predetermined quantity or effective amount of a tricycliccompound of the present invention to produce the desired effect inassociation with a pharmaceutical carrier. The specification for thedosage unit forms of the invention are dictated by and directlydependent on the particular compound and the particular effect, ortherapeutic response, that is desired to be achieved.

Compounds containing Formula III or Formula IV, or pharmaceuticallyacceptable salts, prodrugs, solvates, or hydrates thereof, can beadministered to a patient (an animal or human) via various routesincluding parenterally, orally or intraperitoneally. Parenteraladministration includes the following routes that are outside thealimentary canal (digestive tract): intravenous; intramuscular;interstitial, intraarterial; subcutaneous; intraocular; intracranial;intraventricular; intrasynovial; transepithelial, including transdermal,pulmonary via inhalation, ophthalmic, sublingual and buccal; topical,including dermal, ocular, rectal, or nasal inhalation via insufflationor nebulization. Specific modes of administration shall depend on theindication. The selection of the specific route of administration andthe dose regimen is to be adjusted or titrated by the clinicianaccording to methods known to the clinician in order to obtain theoptimal clinical response. The amount of compound to be administered isthat amount which is therapeutically effective. The dosage to beadministered to a patient shall depend on the characteristics of thepatient being treated, including for example, but not limited to, thepatient's age, weight, health, and types and frequency of concurrenttreatment, if any, of any other chemotherapeutic agent(s), all of whichis determined by the clinician as one skilled in the art.

Compounds containing Formula III or Formula IV, or a pharmaceuticallyacceptable salt, prodrug, solvate or hydrate thereof, that are orallyadministered can be enclosed in hard or soft shell gelatin capsules, orcompressed into tablets. Compounds also can be incorporated with anexcipient and used in the form of ingestible tablets, buccal tablets,troches, capsules, sachets, lozenges, elixirs, suspensions, syrups,wafers and the like. Compounds containing Formula III or Formula IV canbe in the form of a powder or granule, a solution or suspension in anaqueous liquid or non-aqueous liquid, or in an oil-in-water emulsion.

The tablets, troches, pills, capsules and the like also can contain, forexample, a binder, such as gum tragacanth, acacia, corn starch; gelatingexcipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; a sweetening agent, such as sucrose, lactose orsaccharin; or a flavoring agent. When the dosage unit form is a capsule,it can contain, in addition to the materials described above, a liquidcarrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For example,tablets, pills, or capsules can be coated with shellac, sugar or both. Asyrup or elixir can contain the active compound, sucrose as a sweeteningagent, methyl and propylparabens as preservatives, a dye and flavoring.Any material used in preparing any dosage unit form should bepharmaceutically pure and substantially non-toxic. Additionally, thecompounds of Formula III or Formula IV, or a pharmaceutically acceptablesalt, prodrug, solvate or hydrate of said Formulae, can be incorporatedinto sustained-release preparations and formulations.

The compounds of Formula III or Formula IV, or a pharmaceuticallyacceptable salt, prodrug, solvate or hydrate thereof, can beadministered to the central nervous system, parenterally orintraperitoneally. Solutions of the compound as a free base or apharmaceutically acceptable salt can be prepared in water mixed with asuitable surfactant, such as hydroxypropylcellulose. Dispersions alsocan be prepared in glycerol, liquid polyethylene glycols and mixturesthereof, and in oils. Under ordinary conditions of storage and use,these preparations can contain a preservative and/or antioxidants toprevent the growth of microorganisms or chemical degeneration.

The pharmaceutical forms suitable for injectable use include, withoutlimitation, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It can be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Compounds of the present invention may be contained within, mixed with,or associated with, a suitable (acceptable) pharmaceutical carrier foradministration to a patient according to the particular route ofadministration desired. Suitable or acceptable pharmaceutical carriersrefer to any pharmaceutical carrier that will solubilize the compoundsof the present invention and that will not give rise to incompatabilityproblems, and includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic agents, absorptiondelaying agents, and the like. The use of such suitable or acceptablepharmaceutical carriers are well known by those skilled in the art.Preferred carriers include sterile water, physiologic saline, and fivepercent dextrose in water. Examples of other suitable or acceptablepharmaceutical carriers include, but are not limited to, ethanol, polyol(such as propylene glycol and liquid polyethylene glycol), suitablemixtures thereof, or vegetable oils. The proper fluidity can bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size (in the case of adispersion) and by the use of surfactants. The prevention of the actionof microorganisms can be brought about by various antibacterial andanti-fungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.

Sterile injectable solutions are prepared by incorporating the compoundof Formula III or Formula IV in the required amount in the appropriatesolvent with various of the other ingredients enumerated above, asrequired, followed by filtered sterilization. Generally, dispersions areprepared by incorporating the sterilized compound of Formula III orFormula IV into a sterile vehicle that contains the basic dispersionmedium and any of the other ingredients from those enumerated above. Inthe case of sterile powders for the preparation of sterile injectablesolutions, the preferred methods of preparation are vacuum drying andfreeze drying.

Pharmaceutical compositions which are suitable for administration to thenose and buccal cavity include, without limitation, self-propelling andspray formulations, such as aerosol, atomizers and nebulizers.

The therapeutic compounds of Formula III and Formula IV, as describedherein, can be administered to a patient alone or in combination withpharmaceutically acceptable carriers or as pharmaceutically acceptablesalts, solvates or hydrates thereof, the proportion of which isdetermined by the solubility and chemical nature of the compound, chosenroute of administration to the patient and standard pharmaceuticalpractice.

The present invention is more particularly described in the followingnon-limiting examples, which are intended to be illustrative only, asnumerous modifications and variations therein will be apparent to thoseskilled in the art.

EXAMPLES

FIG. 1 shows flow cytometric analysis to assess the effect of variousbicyclic compounds of the present invention on the cell cycle phasedistributions of MDA MB 435 human breast cancer. The percentage of cellsin the G₂/M phases were increased approximately two-fold by treatment ofthe cells for twenty four hours with each of the bicyclic compoundsAAG1, AAG12, AAG16, and AAG20, with AAG1 and AAG16 being most potentbased upon the 30 nM and 10 nM doses, respectively.

FIG. 2 shows the microtubule depolymerization immunofluorescence assayof an A-10 rat smooth muscle cell line following treatment with variouscompounds of the present invention, namely, AAG1, AAG7, and AAG16. A-10rat smooth muscle cells were used since they grow as flat monolayersthat are amenable to imaging. The A-10 cells were treated for twentyfour (24) hours (h) with EtOH (control), 250 nM (nanomolar) AAG1, 500 nMAAG7, or 40 nM AAG16, respectively. Microtubules were then visualized byindirect immunofluorescence staining with beta-tubulin antibodies. Thecontrol cells shown in FIG. 2 displayed extensive microtubule systemswith perimeter organizing centers. Treatment with AAG1, AAG7, and AAG16caused losses of microtubules in the cells. This immunofluorescenceassay shows that the bicyclic compounds of the present invention wereeffective in depolymerizing the tubulin protein microtubule of A-10cells. Each of the compounds AAG1, AAG7, and AAG16 has potent nanomolartubulin inhibitory activity. Compounds of the present invention havingthe structural Formulas III and IV, as set forth herein, inhibit themicrotubule dynamics. The inhibition of microtubule dynamics hindersmicrotubule formation and results in mitotic arrest and initiation ofapoptosis or programmed cell death.

The biological effects of various bicyclic compounds of the presentinvention, namely AAG1, AAG7, AAG12, AAG16, AAG20, and AAG26, ascompared to known antimitotic agents Taxol® (Bristol-Myers SquibbCompany) and combrestastatin A4, commercially available from CaymanChemicals, Michigan, USA, are presented in FIG. 4. Antimitotic compoundsAAG1, AAG7, AAG12, AAG16, AAG20, and AAG26, Taxol®, andcombrestastatinA4, were evaluated for cytotoxity towards the panel ofhuman cell lines MDA MB 435 (human breast cancer), SKOV3 (human ovariancancer), and SKOV3M6/6 (Pgp infected human ovarian cancer). FIG. 4 showsthe IC₅₀ of each of these antimitotic compounds towards each cancer cellline. The IC₅₀ is the inhibitory concentration required to effectuatefifty percent inhibition of cell growth. FIG. 4 shows that the compoundsof the present invention, AAG1, AAG7, AAG12, AAG16, AAG20, and AAG26,have cytotoxic activity toward each of the human cancer cell linestested. Although Taxol® and combrestastatin A4 were more potent thancompounds AAG1, AAG7, AAG12, AAG16, AAG20, and AAG26 in the MDA MB 435and the SKOV3 sensitive cell lines, Taxol® was subject to tumorresistance due to the overexpression of P-glycoprotein (Pgp) in theovarian cancer cell line SKOV3M6/6. FIG. 4 shows the IC₅₀ values of 171nanoM (nM) for AAG1 and 4.4 microM (μM) for Taxol® toward the Pgpinfected human ovarian cancer cell line SKOV3M6/6. FIG. 4 shows thecalculated relative resistance value of 4.7 for compound AAG1 and arelative resistance value of 2013 for Taxol®. FIG. 4 shows the IC₅₀values of 8.4 nM and 3.2 nM for AAG16 and AAG26, respectively, and 4.4microM for Taxol® toward the Pgp infected human ovarian cancer cellline. Thus, the results confirm that overexpression of Pgp did noteffect cell sensitivity to compounds of the present invention AAG3 ofthe present invention.

FIGS. 5 a and 5 b show the results of testing compound AAG1 of thepresent invention using National cancer Institute (NCl) 55 human tumorlines. The cells lines, which represent leukemia, non-small cell lungcancer, colon cancer, central nervous system cancer, melanoma, ovariancancer, renal cancer, prostate cancer, and breast cancer, are listed inFIG. 5. Testing was in accordance with the NCI DevelopmentalTherapeutics Program (DTP) In Vitro Cell Line Screening Project(IVCLSP). Methodology for testing under IVCLSP is provided athttp://dtp.nci.gov/branches/btb/ivclsp.html.

FIG. 5 shows the tumor cell inhibitory activity, measured by GI₅₀ values(10⁻⁸ M) for AAG1. GI₅₀ is the concentration of chemical required toreduce the growth of treated cells to half that of untreated cells (i.e.control). GI₅₀ represents the concentration of chemical required toeffectuate fifty percent inhibition of cell growth. AAG1 exhibited GI₅₀values of single digit 10⁻⁸ molar levels against all 55 tumor celllines.

An NCI COMPARE analysis was performed for AAG1 to elucidate a possiblemechanism of action by comparing responses of the 55 cell lines to knownmicrotubule-targeting agents. For microtubule specific compounds, thecell type selectivity profile in tumor growth inhibitory (TGI) levels ishighly indicative of the compound's mechanism of action. A TGICorrelation value that is equal to or greater than 0.6 is generallyconsidered by those skilled in the art to be a good correlation valuefor classification as a microtubule targeting agent. The results of theNCI COMPARE analysis for compound AAG1 of the present invention is setforth in Table 1.

TABLE 1 TGI endpoint TARGET SET: STANDARD_AGENTS_TGI SEED: S747157 -4MTGI 2 days AVGDATA SEED TYPE: NSC_FIVE_DOSE Rank Vector Correlation Cellline 1 vincristine sulfate S67574 -3M TGI 0.600 49 2 days AVGDATA 2maytansine S 153858 -4M TGI 0.494 49 2 days AVGDATA 3 vinblastinesulfate S49842 -5.6M TGI 0.458 49 2 days AVGDATA 4 homoharringtonineS141633 -4.6M TGI 0.455 47 2 days AVGDATA

The NCI COMPARE analysis was performed for AAG1 to elucidate a possiblemechanism of action of AAG1 by the similarity response of the cell linesto known compounds. The three compounds that showed the best correlationwith AAG1 are all well-known microtubule targeting agents. Formicrotubule specific compounds, the cell type selectivity profile in TGIlevel (correlation) is highly indicative of the compounds mechanism ofaction. Thus AAG1 is a microtubule inhibitor. This COMPARE analysis alsoindicates that AAG1 acts most like vincristine sulfate (correlation0.6), which is a well known anticancer agent widely used in the clinicand strongly suggests that AAG1 would be highly active in vivo. Thetumor inhibitory data from the NCI preclinical tumor screen alsostrongly suggest in vivo activity for AAG1.

FIGS. 6 a, 6 b, and 6 c show individual dose response curves ofpercentage growth for each of the cancer cell lines set forth in FIGS. 5a and 5 b.

FIG. 7 shows a dose response curve of percentage growth for all of thecell lines shown in FIGS. 5 a and 5 b.

FIG. 8 shows mean graphs for each of the cancer types and correspondingcell lines shown in FIGS. 5 a and 5 b.

SYNTHESIS OF BICYCLIC COMPOUNDS Experimental Section

Analytical samples were dried in vacuo (0.2 mm Hg) in a CHEM-DRY dryingapparatus over P₂O₅ at 80° C. Melting points were determined on aMEL-TEMP II melting point apparatus with FLUKE 51K/J electronicthermometer and are uncorrected. Nuclear magnetic resonance spectra forproton (¹H NMR) were recorded on a Bruker WH-400 (400 MHz) spectrometer.The chemical shift values are expressed in ppm (parts per million)relative to tetramethylsilane as an internal standard: s, singlet; d,doublet; t, triplet; q, quartet; m, multiplet; br, broad singlet.Thin-layer chromatography (TLC) was performed on Whatman Sil G/UV254silica gel plates with a fluorescent indicator, and the spots werevisualized under 254 and 366 nm illumination. Proportions of solventsused for TLC are by volume. Column chromatography was performed on a230-400 mesh silica gel (Fisher, Somerville, N.J.) column. Elementalanalyses were performed by Atlantic Microlab, Inc., Norcross, Ga.Element compositions are within ±0.4% of the calculated values.Fractional moles of water or organic solvents frequently found in someanalytical samples of antifolates could not be prevented in spite of24-48 h of drying in vacuo and were confirmed where possible by theirpresence in the ¹H NMR spectra. All solvents and chemicals werepurchased from Aldrich Chemical Co. or Fisher Scientific and were usedas received.

Synthesis of AAG1

Chemistry:

Dimethyl propargylmalonate 1 reacted with acetamidine hydrochloride 2and sodium metal in MeOH (anhydrous) at reflux to cyclize the pyrimidinering and give 3. The use of anhydrous solvent was critical for thesuccess of this reaction. Under cyclization conditions in H₂SO₄ (conc.),compound 3 was converted to the furo[2,3-d]pyrimidine 4, which gave the4-chloro analogue 5 with POCl₃. Compound 5 reacted with N-methyl aniline6 and a trace amount of HCl in BuOH to give AAG1.

Experimental Section for Scheme 12-Methyl-5-prop-2-yn-1-ylpyrimidine-4,6-diol (3)

To a 250 mL flask was added 1 (3.96 g, 20 mmol), 2 (1.85 g, 20 mmol) and50 mL anhydrous MeOH. After 800 mg (20 mmol) Na was added to thesolution, yellow precipitate was observed. The resulting mixture wasrefluxed overnight. The yellow precipitate was collected by filtrationand then dissolved in 10 mL H₂O. The pH of the resulting solution wasadjusted to 6.5 by adding 2 N HCl to afford a yellow precipitate, whichwas collected by filtration and dried over P₂O₅ to afford 1.21 g (37%)of 3: TLC R_(f) 0.11 (CHCl₃/MeOH 6:1); mp>300° C.; ¹H NMR (DMSO-d₆) δ2.23 (s, 3H), 3.05 (s, 2H), 3.32 (s, 1H), 11.92 (s, 2H).

2,6-Dimethylfuro[2,3-d]pyrimidin-4(3H)-one (4)

To a 50 mL flask was added 3 (1.64 g, 10 mmol) and 15 mL H₂SO₄ (conc.).The solution was stirred overnight and poured in to 100 mL distilledwater and extracted by 3×30 mL CHCl₃. The organic layer was pooled andconcentrated to afford 1.36 (83%) of 4 as a yellow powder: TLC R_(f)0.35 (CHCl₃/MeOH 6:1); mp>300° C.; ¹H NMR (DMSO-d₆) δ 2.42 (s, 3H), 2.44(s, 3H), 6.63 (s, 1H), 12.50 (s, 1H).

4-Chloro-2,6-dimethylfuro[2,3-d]pyrimidine (5)

To a 50 mL flask was added 4 (1.64 g, 10 mmol) and 10 mL POCl₃. Theresulting mixture was refluxed for 2 h, and the solvent was removedunder reduced pressure to afford a dark residue. To this was added 30 mLof CHCl₃ and 3 g of silica gel. The solvent was evaporated to afford aplug. Column chromatography of the plug with hexane: acetyl acetate=20:1as eluent afford 1.55 g (85%) of 5 as a yellow solid: TLC

R_(f) 0.26 (Hexane/EtOAC 15:1); mp 47.6-48.1° C.; ¹H NMR (DMSO-d₆) δ2.48 (s, 3H), 2.63 (s, 3H), 6.77 (s, 1H).

N-(4-methoxyphenyl)-N,2,6-trimethylfuro[2,3-d]pyrimidin-4-amine (AAG1)

To a 50 mL flask was added 5 (91 mg, 0.5 mmol), 6 (77 mg, 0.55 mmol) and5 mL BuOH. To this solution was added 2 drops of concentrate HClsolution and the mixture was refluxed. TLC indicated the disappearanceof starting material 5, the solvent was removed under reduced pressure.To the residue obtained was added silica gel and MeOH and the solventremoved to make a plug. This plug was separated by column chromatographyto give 106 g (75%) of AGG1 as a white powder: TLC R_(f) 0.36(Hexane/EtOAC 3:1); mp 108-109° C.; ¹H NMR (DMSO-d₆) δ 2.14 (s, 3H),2.45 (s, 3H), 3.43 (s, 3H), 3.81 (s, 3H), 4.55 (s, 1H), 7.04 (d, 2H,J=2.8 Hz), 7.25 (d, 2H, J=2.8 Hz). Anal. (C₁₆H₁₇N₃O₂) m/z (ESI) 284.1387[M+1]⁺.

Synthesis of AAG 7

Chemistry

Compound AAG 7 was synthesized from the commercial available3-methyladipic acid 1 (Scheme 2). At reflux in concentrated sulfuricacid in ethanol and cyclization in the presence of sodium in toluene, 1further reacted with guanidine carbonate to afford 2 (34%). Chlorinationof 2 to form 3 (34%) was performed by heating with POCl₃ for 3 h.Nucleophilic substitution of 3 with N-methyl-4-methoxyl aniline iniso-propanol gave compound 4 (55%) as a white solid. Compound AAG 7. HClwas precipitated as a white solid when AAG 7 was dissolved in anhydrousether followed by bubbling with anhydrous hydrochloric acid gas (65%).

Experimental Section for Scheme 22-Amino-6-methyl-3,5,6,7-tetrahydro-4H-cyclopenta[d]pyrimidin-4-one (2)

3-Methyladipic acid (1.60 g, 10 mmol) was heated under reflux inethanol/conc. sulfuric acid solution (35 mL, v/v=2.5/1) for 8 h. Thesolution was neutralized with ammonium hydroxide to pH=7, then dilutedwith ethyl acetate (100 mL) and washed with water. The organic phase wasdried with anhydrous sodium sulfate and evaporated to afford a lightyellow liquid which was used in the next step without furtherpurification. The resulting liquid was diluted in anhydrous toluene (100mL) and sodium (0.23 g) was added to the solution in part. The mixturewas heated under reflux for 3 h and cooled, neutralized with 1Nhydrochloric acid solution and washed with water. After drying withanhydrous sodium sulfate, the organic phase was evaporated to afford alight brown liquid. The liquid was used in the next step without furtherpurification. The light brown liquid was diluted with t-BuOH. Guanidinecarbonate (2.70 g, 15 mmol) and potassium tert-butoxide (1.68 g, 15mmol) were added, and the mixture was heated under reflux overnight. Thereaction mixture was cooled, and a precipitate was filtered. The residuewas washed with warm methanol twice (30 mL x1, 15 mL x1). The filtrateand washings were combined and evaporated under reduced pressure, andthe residue was purified by column chromatography usingchloroform/methanol (100/1) as eluent to afford 230 mg of 3 (19% yieldtotal for 3 steps) as white solid. TLC R_(f) 0.36 (CHCl₃/CH₃OH, 10:1);mp: 319-321° C. ¹H NMR (DMSO-d₆): δ 1.10-1.12 (d, 3H, CH₃), δ 1.35-1.46,1.99-2.20, 2.38-2.72, 2.92-2.98 (m, 5H, CH₂CHCH₂), δ 6.32 (br, 2H, NH₂,exch), δ 10.47 (br, 1H, OH, exch). Anal. (C₈H₁₁N₃O. 0.1 CH₃OH)C, H, N:calcd, 57.77, 6.82, 24.95. found, 57.92, 6.78, 24.93.

4-Chloro-6-methyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-2-amine (3)

Compound 2 (297 mg, 1.8 mmol) and phosphorus oxychloride (15 mL) wereheated to reflux for 3 h. The reaction mixture was cooled and evaporatedat reduced pressure, and the residue was diluted with chloroform (50 mL)and neutralized with ammonium hydroxide slowly in an ice bath. Theorganic portion was washed with water (3×30 mL). Solvents wereevaporated at reduced pressure, and the residue was purified by columnchromatography using chloroform/hexane (4/1) as eluent to afford 112 mg(34%) of 3 as a white-off solid. TLC R_(f) 0.48 (CHCl₃/CH₃OH, 10:1); mp:181.5-182.9° C. ¹H NMR (DMSO-d₆): δ 1.16-1.18 (d, 3H, CH₃), δ 1.55-1.65,2.15-2.38, 2.49-2.62, 2.83-2.92 (m, 5H, CH₂CHCH₂), δ 6.83 (br, 2H, NH₂,exch). Anal. (C₈H₁₀ClN₃) C, H, N, Cl: calcd, 52.32, 5.49, 22.88, 19.31.found, 52.33, 5.62, 22.63, 19.09.

N⁴-(4-Methoxyphenyl)-N⁴,6-dimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidine-2,4-diamine(AAG 7)

Compound 3 (94 mg, 0.51 mmol) and N-methyl-4-methoxylaniline (84 mg,0.61 mmol) were dissolved in iso-propanol (5 mL). 37% Hydrochloric acid(2 drops) were added to the solution. The mixture was heated to refluxfor 3 h. Then the reaction was cooled and evaporated at reducedpressure. The residue was diluted with chloroform, neutralized withammonium hydroxide in an ice bath, and then washed with water (2×30 mL).Solvents were evaporated and after drying with anhydrous sodium sulfateand evaporation, the residue was purified by column chromatography usingchloroform as eluent to afford 80 mg of AAG 7 (55%) as a white solid.TLC R_(f) 0.26 (CHCl₃/CH₃OH, 10:1). mp: 146.2-147.5° C. ¹H NMR(DMSO-d₆): δ 0.79-0.81 (d, 3H, CH₃), 81.27-1.35, 1.75-1.89, 2.01-2.10,2.54-2.63 (m, 5H, CH₂CHCH₂), δ 3.25 (s, 3H, NCH₃), δ 3.75 (s, 3H, OCH₃),δ 5.90 (br, 2H, NH₂, exch), δ 6.90-6.92, 7.09-7.11 (dd, 4H, ph-H). Anal.(C₁₆H₂₀N₄O)C, H, N: calcd, 67.58, 7.09, 19.70. found, 67.45, 7.16,19.48.

2-Amino-N-(4-methoxyphenyl)-N,6-dimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-aminiumchloride (AAG 7. HCl)

Compound AAG 7 (150 mg from column) was dissolved in anhydrous ether (25mL) and anhydrous hydrochloric acid gas was bubbled in till no moresolid precipitated out. After filtration, the target compound wasobtained as a white solid (110 mg, 65%). mp: 232.8-233.4° C. ¹H NMR(DMSO-d₆): δ 0.82-0.84 (d, 3H, CH₃), δ 1.23-1.31, 1.78-1.81, 2.20-2.30,2.83-2.89 (m, 5H, CH₂CHCH₂), δ 3.40 (s, 3H, NCH₃), δ 3.79 (s, 3H, OCH₃),δ 6.99-7.01, 7.29-7.31 (dd, 4H, ph-H), δ 7.70 (br, 2H, NH₂, exch), δ12.93 (br, 1H, HCl, exch). Anal. (C₁₆H₂₁N₄OCl. 0.6CH₃OH)C, H, N, Cl:calcd, 58.63, 6.94, 16.48, 10.43. found, 58.58, 6.70, 16.59, 10.41.

Synthesis of AAG11 and AAG12

Experimental Section for Scheme 3

Diethyl {[(E/Z)-2-cyano-1-methylvinyl]amino}malonate (5).

To a suspension of E/Z mixture of 3-aminobut-2-enenitrile 3 (3 g, 35.1mmol) in MeOH (60 mL) was added diethyl aminomalonate hydrochloride 4(7.9 g, 36.8 mmol). The resulting mixture was stirred at roomtemperature for 5 h. TLC showed the disappearance of the startingmaterials and the formation of one major spot at R_(f) 0.26 (ethylacetate/n-hexane, 1:2). The reaction solvent was diluted with ethylacetate (50 mL), washed with brine (30 mL×2), dried over MgSO₄. To theorganic solvent 15 g silica gel was added and the mixture was evaporatedto dryness under reduced pressure. This silica gel plug was loaded on adry silica gel column (2×15 cm) and flash chromatographed initially withn-hexane (200 mL), then sequentially with 500 mL 5% ethyl acetate inn-hexane, 500 mL 10% ethyl acetate in n-hexane, 500 mL 15% ethyl acetatein n-hexane. Fractions containing the desired product (TLC) were pooledand evaporated to afford 6.74 g (80%) of 5 as an off-white solid: mp50-52° C.; ¹H NMR (DMSO-d₆) δ 1.18-1.23 (t, 3H, J=6.9 Hz), 2.05 (s, 6H),E isomer 3.94 (s, 1H), 4.18-4.24 (m, 4H), 4.96 (d, 1H, J=7.8 Hz), Zisomer 5.32 (s, 1H), 7.51-7.56 (d, 2H, J=7.8 Hz). Anal. (C₁₁H₁₆N₂O₄) C,H, N.

Ethyl 3-amino-5-methyl-1H-pyrrole-2-carboxylate (6)

A solution of NaOEt in EtOH (0.5 M, 120 mL) was added slowly to astirred solution of 5 (1 g, 4 mmol) in 20 mL EtOH. The reaction mixturewas stirred for 6 h at 60° C. and cooled to room temperature; thesolvent was evaporated under reduced pressure. The crude product waspurified by column chromatography on silica gel with 10% ethylacetate/n-hexane as the eluent to yield 6 (0.45 g, 65%) as an off-whitesolid: mp 85-87° C.; R_(f) 0.36 (ethyl acetate/n-hexane, 1:1); ¹H NMR(DMSO-d₆) δ 1.24 (t, 3H, J=6.4 Hz), 2.03 (s, 3H), 4.12 (q, 2H, J=6.4Hz), 4.91 (s, 2H), 5.26 (s, 1H), 10.21 (s, 1H). Anal. (C₈H₁₂N₂O₂) C, H,N.

Methyl(6-methyl-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-2-yl)carbamate(7)

The pyrrole 6 (2.68 g, 16 mmol) was dissolved in MeOH (40 mL), and1,3-bis(methoxycarbonyl)-2-methyl-2-thiopseudourea (3.74 g, 18 mmol) wasadded followed by AcOH (4.6 mL). The mixture was stirred at roomtemperature overnight and became a thick paste. To the reaction mixtureNaOMe in MeOH (25%) 45 mL was added, and stirring was continued at roomtemperature for 2 h. The mixture was neutralized with AcOH and the solidcollected by filtration and washed well with water. After drying, 7(2.44 g, 69%) was obtained as an off-white powder: mp 234-236° C.; TLCR_(f) 0.22 (MeOH/CHCl₃, 1:5); ¹H NMR (DMSO-d₆) δ 2.28 (s, 3H, CH₃), 3.73(s, 3H, OCH₃), 5.95 (s, 1H), 10.90 (s, 1H), 11.10 (s, 1H), 11.76 (s,1H). Anal. (C₉H₁₀N₄O₃′0.79C₆H₆.0.55C₇H₈O₃S)C, H, N.

2-Amino-6-methyl-3,5-dihydro-4H-pyrrolo[3,2-d]pyrimidin-4-one (8)

To a 200 mL round bottomed flask was added 7 (1 g, 4.5 mmol) suspendedin 1N NaOH (35 mL). The reaction mixture was heated at 55° C. for 3 h.The resulting solution was cooled in an ice bath and neutralized withAcOH. The precipitated solid was collected by filtration, washed withbrine, and dried in vacuo to afford 0.67 g (92%) of 8 as a white solid:mp 252-254° C.; TLC R_(f) 0.15 (MeOH/CHCl₃, 1:5); ¹H NMR (DMSO-d₆) δ2.20 (s, 3H), 5.65 (s, 1H), 5.65 (s, 2H), 10.21 (s, 1H), 11.15 (s, 1H).Anal. (C₇H₈N₄O.0.73H₂O)C, H, N.

2,2-Dimethyl-N-(6-methyl-4-oxo-4,5-dihydro-3H-pyrrolo[3,2-d]pyrimidin-2-yl)propanamide(9)

To a 250-mL round-bottomed flask was added 8 (1.37 g, 8 mmol) suspendedin 40 mL of dichloroethane; then trimethylacetyl chloride (1.99 mL, 16mmol), DMAP (0.13 g, 1 mmol) and triethylamine (2.68 mL) were added. Themixture was stirred overnight at 50° C. The resulting mixture wascooled, diluted with dichloromethane (50 mL), washed with brine (40mL×2), dried over Na₂SO₄ and concentrated in vacuo. To this solutionwere added methylene chloride (30 mL) and silica gel (5 g) and thesolvent evaporated. The silica gel plug obtained was loaded onto asilica gel column and eluted with 9:1 ethyl acetate/n-hexane Thefractions containing the product (TLC) were pooled and the solvent wasevaporated to afford 1.33 g (67%) of 9 as a white solid: TLC R_(f) 0.47(MeOH/CHCl₃, 1:10); mp 156-157° C.; ¹H NMR (DMSO-d₆) δ 1.19 (s, 9H),2.29 (s, 3H), 5.97 (s, 1H), 10.72 (s, 1H), 11.78 (s, 1H), 11.87 (s, 1H).Anal. (C₁₂H₁₆N₄O₂″0.15CH₃COCH₃) C, H, N.

N-(4-chloro-6-methyl-5H-pyrrolo[3,2-d]pyrimidin-2-yl)-2,2-dimethylpropanamide(10)

To a 100-mL round-bottomed flask was added 9 (1.16 g, 4.67 mmol)suspended in 30 mL phosphorus oxychloride. The reaction mixture washeated at reflux with stirring in an anhydrous atmosphere for 3 h. Thedark orange solution was allowed to cool to room temperature andconcentrated in vacuo. Water (20 mL) was then added to the residue at 0°C. with vigorous stirring to give an exothermic reaction. Concentratedaqueous ammonium hydroxide was added to pH 5 to give a precipitate,which was collected by filtration, washed with water (3×5 mL), and driedin vacuo. The crude product was purified by silica gel columnchromatography with 2% MeOH/CHCl₃. Recrystallization from MeOH afforded1.07 g (86%) of 10 as a white solid: TLC R_(f) 0.35 (MeOH/CHCl₃, 1:10);mp 162-163° C.; ¹H NMR (DMSO-d₆) δ 1.19 (s, 9H), 2.47 (s, 3H), 6.33 (s,1H), 9.85 (s, 1H), 12.07 (s, 1H). Anal. (C₁₂H₁₅ClN₄O)C, H, N, Cl.

N-(4-methoxy-phenyl)-6-methyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine(AAG11)

To a 100-mL round-bottomed flask, flushed with nitrogen, were added 10(0.2 g, 0.7 mmol), 4-methoxy-phenylamine (0.12 g, 1.05 mmol), i-PrOH (20mL), and 2-3 drops of concd HCl. The reaction mixture was heated atreflux with stirring for 45 min until the starting material 10disappeared (TLC). The reaction solution was allowed to cool to roomtemperature; the solvent was removed under reduced pressure, 1,4-dioxane(10 mL) and 10 mL of 15% KOH aqueous solution were added. The resultingmixture was heated at reflux overnight. After cooling the reactionsolution was neutralized with 1 N HCl, and then evaporated in vacuo todryness and the residue was purified by column chromatography on silicagel with 2% MeOH in CHCl₃ as the eluent. Fractions containing theproduct (TLC) were combined and evaporated to afford 0.1 g (53%) ofAAG11 as a brown solid: mp 201-202° C.; R_(f) 0.42 (MeOH/CHCl₃, 1:5); ¹HNMR (DMSO-d₆) δ 2.34 (s, 3H), 3.78 (s, 3H), 5.33 (s, 2H), 5.75 (s, 1H),6.86 (d, 2H, J=6.3 Hz), 7.72 (d, 2H, J=6.3 Hz), 8.45 (s, 1H), 8.52 (s,1H). Anal. (C₁₄H₁₅N₅O.0.71CHCl₃′0.81HCl) C, H, N.

N-(4-methoxy-phenyl)-6-methyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine(AAG12)

(synthesized as described for AAG11): yield 47%; TLC R_(f) 0.48(MeOH/CHCl₃, 1:5); mp 161-163° C.; ¹H NMR (DMSO-d₆) δ 2.12 (s, 3H), 3.37(s, 3H), 3.78 (s, 3H), 5.31 (s, 2H), 5.70 (s, 1H), 6.96 (d, 2H, J=5.4Hz), 7.15 (d, 2H, J=5.4 Hz), 8.16 (s, 1H). Anal. (C₁₅H₁₇N₅O″0.78H₂O)C,H, N.

Synthesis for AAG16

Synthesis of AAG16 is shown in Scheme 6 above. Compound 3 was obtainedas per reported method by stirring 1 with 2 at room temperature in thepresence of triethylamine and was used without purification for furthersteps. Compound 3 was reacted with free base of formamidinehydrochloride 4 (obtained by stirring at room temperature with sodiumethoxide) in ethanol at reflux to obtain furo[2,3-d]pyrimidine 5.Interestingly no rearrangement to pyrrolo[2,3-d]pyrimidine as before wasobserved. The product 5 was confirmed using ¹HMNR and elementalanalysis. The amino group of 5 was coupled with 6 using copper (I)iodide and L-proline as a chelating ligand in the presence of potassiumcarbonate in DMF to afford 7. Compound 7 was N-methylated by treating itwith sodium hydride followed by dimethyl sulfate to obtain AAG16.

Experimental Section for Scheme 4 5-methylfuro[2,3-d]pyrimidin-4-amine(5)

Sodium metal (2.3 g; 0.1M) was added cautiously to stirred anhydrousEthanol (5.8 mL, 0.1M) over 10 min at room temperature. After stirringthe resulting slurry for additional 5 min, 4 (8.05 gm, 0.1M) was added.The slurry was stirred at room temperature for 30 min after whichsolution of 3 (13 g crude; ≈0.1M) in anhydrous ethanol (200 mL) wasadded. The mixture was heated at reflux for 8 h. After cooling thereaction mixture to room temperature, silica gel (25 g) was added andsolvents evaporated under reduced pressure to obtain a plug.Purification was done by flash chromatography using 1% methanol inchloroform. The fractions corresponding to the product spot were pooledand evaporated under reduced pressure to obtain 5 (5.3 g, 35%) aslustrous pink crystals. TLC R_(f) 0.29 (CHCl₃: MeOH, 10:1); mp240.2-242.5° C.; ¹H NMR (300 MHz) (DMSO-d₆): δ 2.28 (s, 3H, CH₃); 7.018(br, 2H, NH₂, exch), 7.528 (s, 1H, C6-CH), 8.12 (s, 1H, C2-CH). Anal.Calcd for C₇H₇N₃O: C, 56.37; H, 4.73; N, 28.17. Found: C, 56.48; H,4.74; N, 28.17.

N-(4-methoxyphenyl)-5-methylfuro[2,3-d]pyrimidin-4-amine (7)

A 50 mL round bottom flask with a stir bar was charged with copperiodide (66.5 mg, 0.35 mmol), anhydrous potassium carbonate (480 mg, 3.5mmol), L-proline (80 mg, 0.7 mmol), 5 (150 mg, 1 mmol) and 6 (350 mg,3.5 mmol). The flask was connected to vacuum for 3 min followed by theaddition of anhydrous DMF (15 mL) using syringe. The flask was purgedwith argon for 5 min and then heated in an oil bath maintained at 110°C. On heating the suspension became bluish grey which lasted for about 2h. The reaction was stirred for additional 22 h at 110° C. at the end ofwhich the mixture was allowed to cool to room temperature. Ethyl acetate(25 mL) was added and the mixture was poured into water (100 mL). Theproduct was extracted with ethyl acetate (100 mL×2). The combinedorganic extracts were washed with brine (100 mL) and dried (anhydroussodium sulfate) and concentrated under reduced pressure. Silica gel (500mg) was added and solvent evaporated to obtain a plug. Purification bycolumn chromatography using hexanes and ethyl acetate (10:1 to 2:1)afforded 7 (140 mg, 56%) as light brown solid. TLC R_(f) 0.77 (CHCl₃:MeOH, 10:1); mp 99-101.6° C.; ¹HNMR (400 MHz) (DMSO-d₆): δ 2.383-2.386(d, 3H, CH₃, J=1.2 Hz); 3.747 (s, 3H, OCH₃), 6.919-6.941 (d, 2H, C₆H₄,J=8.8 Hz), 7.466-7.488 (d, 2H, C₆H₄, J=8.8 Hz), 7.65-7.653 (d, 1H,C6-CH, J=1.2 Hz), 8.234 (s, 1H, C2-CH), 8.381 (s, 1H, 4-NH, exch). Anal.Calcd for C₁₄H₁₃N₃O₂: C, 65.87; H, 5.13; N, 16.46.

Found: C, 65.94; H, 5.13; N, 16.42.

N-(4-methoxyphenyl)-N,5-dimethylfuro[2,3-d]pyrimidin-4-amine (AAG16)

To a 25 mL round bottom flask was weighed 7 (51 mg, 0.2 mmol) and wasadded DMF (2 mL) to afford a solution. The flask was purged with argonfor five min followed by cooling down to 0° C. using ice bath. Sodiumhydride (14.4 mg, 0.6 mmol) was added to the solution at 0° C. Thesolution was stirred for 30 min at 0° C. under argon atmosphere.Dimethyl sulfate (75.7 mg; ≈57 μl, 0.6 mmol) was injected to thereaction mixture and the flask was warmed to room temperature. Themixture was stirred at room temperature for another 3 h at the end ofwhich 1 N Hydrochloric acid (5 mL) was added carefully to quench thereaction followed by water (20 mL) to afford a precipitate. Product wasextracted using ethyl acetate (10 mL×2). Combined organic extracts werewashed with brine (10 mL) dried (anhydrous sodium sulfate) andconcentrated under reduced pressure. Silica gel (200 mg) was added andsolvent evaporated to afford a plug. Column chromatography by elutionwith hexanes:ethyl acetate (5:1) afforded AAG16 (20 mg; 37%) as lightbrown semisolid; which was triturated with hexanes to afford light brownsolid. TLC R_(f) 0.79 (CHCl₃: MeOH, 10:1); mp 84-85.6° C.; ¹HNMR (400MHz) (DMSO-d₆): δ 1.036-1.039 (d, 3H, CH₃, J=1.2 Hz); 3.423 (s, 3H,NCH₃), 3.752 (s, 3H, OCH₃), 6.944-6.967 (d, 2H, C₆H₄, J=9.2 Hz),7.176-7.199 (d, 2H, C₆H₄, J=9.2 Hz), 7.505-7.508 (d, 1H, C6-CH, J=1.2Hz), 8.234 (s, 1H, C2-CH). Anal. Calcd for C₁₅H₁₅N₃O₂.0.28C₆H₁₄.0.05HCl: C, 67.84; H, 6.48; N, 14.22. Found: C, 67.89; H, 6.18; N,14.06.

Synthesis of AAG20

Experimental Section for Scheme 52-Methyl-4-hydroxypyrrolo[2,3-d]pyrimidine (3)

Acetamidine hydrochloride (2, 0.05 mol, 4.7 g) was added to the 0.1Msodium ethoxide solution (75 ml) and kept stirring under roomtemperature for 0.5 h. After removing the formed sodium chloride byfiltration, the filtrate was added the ethylα-cyano-γ,γ-diethoxybutyrate (1, 0.05 mol, 11.5 g) and the solution washeated under reflux for 5 h. After the removal of most solvent undervacuum, acetic acid was added to adjust the pH to 7.0 and 10.8 gprecipitation as white powder. Ethanol (110 ml) with concentratedsulfuric acid (2 ml) was added to the collected powder and was refluxedfor 2 h. By the end of the reaction an equal volume of water was addedand kept at 4° C. overnight. The pyrrolopyrimidine 3 precipitated aswhite powder (2.1 g) was used for next step without furtherpurification.

4-chloro-2-methyl-7H-pyrrolo[2,3-d]pyrimidine (4)

The pyrrolopyrimidine 3 (0.01 mol, 1.50 g) was treated with excess POCl₃(20 ml) under reflux for 2 h. Then the remaining POCl₃ was removed undervacuum followed by chromatography purification to afford 7 as a whitepowder (1.42 g) with a yield of 85%. TLC R_(f)=0.62 (CH₃OH:CHCl₃=1:5).¹H NMR (DMSO-d6): δ 2.61 (s, 3H, 2-CH₃), 6.52-6.53 (dd, 1H, 5-H),7.56-7.58 (dd, 1H, 6-H), 12.30 (s, 1H, 7-H).

N-(4-methoxyphenyl)-N,2-dimethyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine(AAG20)

Compound 4 (0.005 mol, 0.835 g) was added to a solution of4-methoxy-N-methylaniline (5, 0.006 mol, 0.822 g) in i-propanol (10 ml)with drops of conc. HCl. The resulting solution was transferred to amicrowave vial (20 ml) and irradiated at 110° C. After 6 h, the reactionwas completed and AAG20 was obtained after the chromatographicpurification as a white solid (1.01 g) with 80% yield. TLC R_(f)=0.85(CH₃OH:CHCl₃=1:5). ¹H NMR (DMSO-d6): δ 2.65 (s, 3H, 2-CH₃), 3.62 (s, 3H,N—CH₃), 3.84 (s, 3H, OCH₃), 6.99 (bs, 4H, C₆H₄), 7.11-7.13 (d, 1H, 5-H),7.39-7.41 (d, 1H, 6-H), 12.44 (s, 1H, 7-H, D₂O exchanged). Anal. Calcd.(C₁₅H₁₆N₄O. 0.3688 CHCl₃.), C, 59.10; H, 5.28; N, 17.94. Found C, 58.98;H, 5.65; N, 18.16.

Synthesis of AAG 26

Chemistry

Compound AAG 26 was synthesized from the commercial available3-methyladipic acid 1 (Scheme 6). At reflux in concentrated sulfuricacid in ethanol and cyclization in the presence of sodium in toluene, 1further reacted with acetamidine hydrochloride to afford 2 (30%).Chlorination (69%) of 2 with POCl₃ for 3 h afforded 3. Nucleophilicsubstitution of 3 with N-methyl-4-methoxyl aniline in iso-propanolafforded 4, which was further diluted with anhydrous ether and anhydroushydrochloric acid gas was bubbled in to afford AAG 26 as a white solid(50% for two steps).

Experimental Section for Scheme 62,6-Dimethyl-3,5,6,7-tetrahydro-4H-cyclopenta [d]pyrimidin-4-one (2)

3-Methyladipic acid 1 (1.60 g, 10 mmol) was heated under reflux inethanol/conc. sulfuric acid solution (35 mL, v/v=2.5/1) for 8 h. Thesolution was neutralized with ammonium hydroxide to pH=7, then dilutedwith ethyl acetate (100 mL) and washed with water. The organic phase wasdried with anhydrous sodium sulfate and evaporated to afford a lightyellow liquid which was used in the next step without furtherpurification. The resulting liquid was diluted in anhydrous toluene (100mL) and sodium (0.23 g) was added to the solution in part. The mixturewas heated under reflux for 3 h and cooled, neutralized with 1Nhydrochloric acid solution and washed with water. After drying withanhydrous sodium sulfate, the organic phase was evaporated to afford alight brown liquid. The liquid was used in the next step without furtherpurification. The light brown liquid was diluted with t-BuOH.Acetamidine (1.13 g, 12 mmol) and potassium tert-butoxide (1.34 g, 12mmol) were added, and the mixture was heated under reflux overnight. Thereaction mixture was cooled and the precipitate was filtered. Theresidue was washed with warm methanol twice (30 mL x1, 15 mL x1). Thefiltrate and washings were combined and evaporated under reducedpressure, and the residue was purified by column chromatography usingchloroform/methanol (100/1) as eluent to afford 345 mg (21% yield totalfor 3 steps) as a white solid. TLC R_(f) 0.30 (CHCl₃/CH₃OH, 10:1); mp:173.9-175.4° C. ¹H NMR (DMSO-d₆): δ 1.05-1.07 (d, 3H, CH₃), δ 2.24 (s,3H, CH₃), δ 2.13-2.34, 2.73-2.90 (m, 5H, CH₂CHCH₂), δ 12.16 (br, 1H, OH,exch). Anal. (C₉H₁₂N₂O)C, H, N: calcd, 65.83, 7.37, 17.06. found, 65.85,7.36, 17.12.

N-(4-Methoxyphenyl)-N,2,6-trimethyl-6,7-dihydro-5H-cyclopenta[d]pyrimidin-4-aminiumchloride (AAG 26)

Compound 2 (0.25 g, 1.5 mmol) and phosphorus oxychloride (10 mL) wereheated to reflux for 3 h. The reaction mixture was cooled down andevaporated at reduced pressure, and the residue was diluted withchloroform (50 mL) and neutralized with ammonium hydroxide slowly in anice bath. The organic portion was washed with water (3×30 mL). Solventswere evaporated at reduced pressure, and the residue was purified bycolumn chromatography using chloroform/hexane (4/1) as eluent to afford139 mg of 3 as a colorless liquid (69%). Compound 3 (185 mg, 1.01 mmol)and N-methyl-4-methoxylaniline (167 mg, 1.22 mmol) were dissolved iniso-propanol (5 mL). 37% Hydrochloric acid (2 drops) was added in thesolution. The mixture was heated to reflux for 3 h. Then the reactionwas cooled and evaporated at reduced pressure. The residue was dilutedwith chloroform, neutralized with ammonium hydroxide, and then washedwith water (2×30 mL). Solvents were evaporated and after drying withanhydrous sodium sulfate and evaporation, the residue was purified bycolumn chromatography using chloroform as eluent to afford 4 as a lightyellow liquid. TLC R_(f) 0.24 (CHCl₃/CH₃OH, 10:1). The liquid wasdiluted with anhydrous ether (10 mL) and anhydrous hydrochloric acid gaswas bubbled in till no more solid precipitated out. After filtration,the target compound AAG 26 was obtained as a white solid (163 mg, 50%for two steps). mp: 196-198° C. ¹H NMR (DMSO-d₆): δ 0.85-0.86 (d, 3H,CH₃), δ 1.37-1.47, 1.89-1.99, 2.43-2.44, 2.98-3.05 (m, 5H, CH₂CHCH₂), δ2.60 (s, 3H, 2-CH₃), δ 3.51 (s, 3H, NCH₃), δ 3.80 (s, 3H, OCH₃), δ7.01-7.03, 7.33-7.35 (dd, 4H, ph-H), δ 14.88 (br, 1H, HCl, exch). Anal.(C₁₇H₂₂N₃OCl) C, H, N, Cl: calcd, 63.84, 6.93, 13.14, 11.08. found,63.75, 6.88, 13.05, 10.98.

Preferred Substituted Cyclopenta Pryrimidine Compounds Introduction

Tubulin binding agents (see FIG. 9 for known microtubule targetingagents) belong to an important class of antitumor agents and are widelyused in the clinic for cancer chemotherapy. Most microtubule targetingagents can be divided into three classes based on their interactionswithin the taxane, vinca, or colchicine site on tubulin. Drugs that bindwithin the taxoid site include paclitaxel (Taxol), docetaxel (Taxotere),and the epothilones. Paclitaxel and other taxoids (and the epothilones)bind to β-tubulin in the interior of the microtubule. Unlike the othertwo classes of antimicrotubule agents, the taxoids stimulate tubulinpolymerization and are designated as microtubule stabilizers. They areuseful in the treatment of breast, lung, ovarian, head and neck andprostate cancers. The second class of microtubule disrupting agents arethe vinca alkaloids and these include vincristine, vinblastine,vindesine and vinorelbine. The vinca alkaloids bind between twoαβ-tubulin diemers at a site that is distinct from the taxane site. Thevinca alkaloids are important in the treatment of leukemias, lymphomas,non-small cell lung cancer and childhood cancers. A diverse collectionof small molecules, including colchicine and the combretastatins, bindto the colchicine site on β-tubulin at its interface with α-tubulin, asite distinct from the vinca site. Similar to the vinca alkaloids,colchicine site agents inhibit tubulin polymerization. Colchicine itselfis not used as an antitumor agent but is useful in the treatment of goutand familial Mediterranean fever. Although there are no clinicallyapproved anticancer agents that bind within the colchicine site, severalagents including 2-methoxyestradiol, combretastatin A-4 (CA-4)phosphorylated prodrug combretastatin A-4 phosphate (CA-4P)(fosbretabulin), the combretastain CA-1P prodrug (OXi4503), BNC105P,ABT-751 and plinabulin (NPT-2358) have been evaluated in clinicaltrials. While CA-4P, CA-1P and others continue to be evaluated inclinical trials, to date no colchicine site agent has advanced to PhaseIII studies or FDA approval for anticancer indications, demonstratingthe need of developing additional colchicine site agents for potentialclinical evaluations.

Preferred Methoxynaphthyl Substituted Cyclopenta Pyrimidine Compounds:

Compounds 1, 2 are cyclopenta pyrimidine compounds. Their antitubulinactivities are set forth above. Compound 2 is a high potency antitubulinagent (IC₅₀=25.7 nM), while the N-demethyl compound 1 is almostinactive. The structural differences between Compounds 1 and 2 lead toseveral pharmacological ramifications. Among them, the conformation ofaniline rings maybe a pivotal factor that results in the affinitydifference. In order to validate this, we set forth a preferredrepresentative series of N-methyl-N-methoxynaphthylcyclopenta[d]pyrimidin-4-amine (4) based on the high potent antibulinagent 3 (IC₅₀=7.0 nM). All these preferred representativemethoxynaphthyl substituted bicyclic compounds of this invention havethe same function groups and molecular weight, similar Log D.

Chemistry:

Synthesis Scheme of more preferred methoxynaphthyl substitutedcyclopenta pyrimidine compounds 10-14 of this invention:

Formyl protection of the amine in hydroxyaminonaphthalenes 5 with formicacid and acetic anhydride afforded N-formyl hydroxynaphthylamine 6 in90-95% yield. Dimethylation of 6 using NaH and methyl iodide in DMF at0° C. gave N-methyl-N-formyl methoxynaphthylamine 7 in 80-90% yield.Deformylation of 7 in refluxing concentrated HCl afforded N-methylmethoxylnaphthylamine 8 in 90-95% yield. Nucleophilic displacement of4-chlorocyclopenta[d]pyrimidine 9 by secondary amine in compounds 8afforded target more preferred methoxynaphthyl substituted cyclopentapyrimidine compounds 10-14 of this invention.

Biological Evaluation of Preferred Methoxynaphthyl SubstitutedCyclopenta Pyrimidine Compounds 10-14 of this Invention:

TABLE 2 IC₅₀ values for inhibition of proliferation of MDA-MB-435 cellsand effect on microtubule polymerization. EC₅₀ in A-10 cells (TubulinIC₅₀ ± SD depolymerization (MDA-MB-435) inhibitory acitivity) Compd.Tumor cells (nM) (nM) 3 7.0 ± 0.7 25.9 ± 3.7 10 ND >10 000 11 ND >10 00012 33.7 ± 3.4   184 ± 4.8 13 10.2 ± 0.46 14 234 ± 20   500 ± 2.1 CA4 3.4± .6  13.0

Compounds 3, 10-14 were tested for antiproliferative effects against thedrug sensitive MDA-MB-435 tumor cell line in culture using thesulforhodamine B assay reported for the way as standard antitubulinagent CA4. The microtubule disrupting effects of these compounds wereobserved in a cell-based phenotypic screen. The data (Table 2) indicatethat 13 has the comparable antiproliferative effects as CA4 with an IC₅₀of 10 nM. However, the reverse orientation of methoxy and methylamino onthe naphthyl, compound 11, has no microtubule depolymerizationinhibitory activity up to 10 000 nM. Compound 14 is less potent than 13,but more potent than 11.

TABLE 3 Inhibition of tubulin assembly and colchicine binding by CA-4,for more preferred substituted cyclopenta pyrimidine compounds 10-14 ofthis invention: Inhibition of colchicine binding Inhibition of %Inhibition ± SD tubulin assembly 5 μM 1 μM Compd. IC₅₀ (μM) ± SDinhibitor inhibitor 10 >20 (minimal act) 11 >20 (partial act) 12  1.2 ±0.04 93 ± 0.6 77 ± 1   13 0.98 ± 0.04 98 ± 0.3 86 ± 0.7 14  1.4 ± 0.00772 ± 0.6 CA4  1.1 ± 0.1 99 ± 0.6 90 ± 0.2

The effects of designed compounds 10-14 on the polymerization ofpurified tubulin were evaluated. This allows for the study of the directinteraction of the compounds with their intracellular target. Theability of these compounds to bind to the colchicine site on tubulin wasevaluated by measuring inhibition of [³H]colchicine binding to tubulin.The data (Table 3) show that compound 13 is an effective and potentinhibitor of tubulin assembly. It is clear that at 1 and 5 μMconcentrations, 13 inhibited the binding of [³H]colchicine, somewhatmore potently than CA4. It is therefore likely that 13 binds in thecolchicine site. Contrary to 13, 11 only shows partial inhibitoryactivity on tubulin assembly at 20 μM concentration.

TABLE 4 Circumvent clinically relevant models of drug resistance of morepreferred substituted cyclopenta pyrimidine compounds 10-14 of thisinvention: IC₅₀ ± SD (nM) β-III Wild type Overexpressing Compd. HeLaHeLa Rr 10 ND ND 11 >10,000 >10 000 12  15 ± 0   14 ± 1 1.07 13 4.0 ± 1 4.0 ± 1 1.0 14  85 ± 5  180 ± 40 0.47 CA4 1.8 ± 0.4  2.5 ± 0.7 0.72Paclitaxel 5.3 ± 2   16 ± 1 0.33 Docetaxel 4.0 ± 2   13 ± 4 0.31

One mechanism of drug resistance that can lead to chemotherapy failurewith tubulin targeting agents is the expression of the βIII isotype oftubulin. An isogenic HeLa cell line pair was used to evaluate theeffects of βIII tubulin expression on the activities of 10-14 incomparison with paclitaxel, Docetaxel and CA4. 13, and CA4 had Rr of1.0, 0.72, respectively (Table 4), in this cell line pair, suggestingthat, like 13, they overcome drug resistance mediated by βIII tubulincompared with Paclitaxel and Docetaxel, which had a Rr of 0.33 and 0.31.These results suggest that 13 would circumvent tumor resistance becauseof the overexpression of βIII tubulin.

Conformation Study:

FIGS. 10, 11, and 12 show the results of the NOESY study of the morepreferred substituted cyclopenta pyrimidine compounds 11, 13 and 14 ofthis invention, respectively, and spectrum interpretation.

The 1-D ¹HNMR and 2-D ¹HNMR (NOESY) spectrum study, in DMSO-d₆ and D₂O,was carried out to explore the stable conformation of the more preferredsubstituted cyclopenta pyrimidine compounds 11, 13 and 14 of thisinvention. In 1-D ¹HNMR, the chemical shift of 5-position protons in allthree compounds shifts to upfield (less than 2 ppm), which indicatesthat the 5-position protons of cyclopenta[d]pyrimidine reside atshielding cone of the naphthyl ring. In 13, the chemical shift of two5-position protons is identical, which illustrates these two protonsreside at the exact same environment. In 11 and 14, the two 5-positionprotons have different chemical shift, which suggest one atropisomer hashigher population than the other atropisomer. Due to 8′-H, the rotationability of naphthyl ring around cyclopenta[d]pyrimidine ring incompounds 11 and 14 is somewhat restrained.

NOESY is a useful tool to illustrate the space relationship of protonsthat have distance between 1.8-4.3 angstrum. The 2-D ¹HNMR (NOESY)spectrum of 13, both of 5-position protons in NOE effect range to both1′ and 3′ protons on the naphthyl ring, while the distance of 5-positionprotons and N-methyl protons is beyond the NOE effect range. This datasuggest that napthyl ring is hanging above the cyclopenta ring, and theatropisomers, if exist, are fast interchangeable due to the freelyrotatable σ-bonds (C₁—N and N—C₂′). In compound 11, one of 5-positionproton (5a) residues in the NOE effect range of 8′ proton, but not 2′proton. The other 5-position proton (5b) residues in the NOE effectrange of 2′ proton, but not 8′ proton. This data suggest that 11 adoptsone atropisomer over the other thanks to the absent of free rotation ofnaphthyl ring toward the cyclopenta[d]pyrimidine ring, in the NMR tookplace environment (room temperature, DMSO & D₂O solution). In compound14, the two atropisomers is partially convertible. It is clear thenaphthyl rings of 11, 13 and 14 place different orientation towardscyclopenta[d]pyrimidine rings, although all the naphthyl rings hangabove the cyclopenta side.

FIGS. 13 a and b show MOE (2012) simulation of the low energy conformerof the more preferred substituted cyclopenta pyrimidine compounds 11 and13 of this invention, respectively. Only the atropisomer thatmethoxynaphthyl group point to the left side were showed and theatropisomer with methoxynapthyl group point to the right side does notpresent. FIG. 13 a: Up-left: the distances of 5-position protons and 2′and 8′ protons of 11 in low energy conformer. Distances are: 5a-2′ (4.30Å); 5a-8′ (3.35 Å); 5b-2′ (3.24 Å); 5b-8′ (4.51 Å), which are generallymatching with the NOESY results. Up-right: the orthogonal angle of thisconformer is 101.7°. FIG. 13 b: Low-left: the distances of 5-positionprotons and 1′ and 3′ protons of 13 in low energy conformer. Distancesare: 5a-1′ (3.73 Å); 5a-3′ (2.72 Å); 5b-1′ (3.80 Å); 5b-8′(4.70 Å),which are matching with the NOESY results. Low-right: the orthogonalangle of this conformer is 105.2°.

In order to establish the orthogonal angle of low energy conformer, theatropisomer (FIG. 13) with methoxynaphthyl point to the left of the morepreferred substituted cyclopenta pyrimidine compounds 11 and 13 werechosen to run an energy minimization (MOE, 2012). In 11, distances of5-position protons and 2′ and 8′ protons are: 5a-2′ (4.30 Å); 5a-8′(3.35 Å); 5b-2′ (3.24 Å); 5b-8′ (4.51 Å), which are generally matchingwith the NOESY results. The orthogonal angle of naphthyl ring tocyclopenta[d]pyrimidine ring is 101.7°. In 13, distances of 5-positionprotons and 1′ and 3′ protons are: 5a-1′ (3.73 Å); 5a-3′ (2.72 Å); 5b-1′(3.80 Å); 5b-8′(4.70 Å), which again are matching with the NOESYresults. The orthogonal angle of naphthyl ring tocyclopenta[d]pyrimidine ring is 105.2°. Further, we have carried outsuperimposition of 11 and 13 docked in the colchicine site of tubulin,which indicate that the naphthyl is involved in the π-stack interactionwith Tyr224 and that the methoxy is involved in the H-B with ammonium ofLys254.PDB:1SA0. A docking study of compounds 11 and 13 into colchicinesite of tubulin has been carried out to elucidate the relationship ofthe orthogonal angle with binding affinity. The result suggests that themethoxynaphthyl in 13 has stronger it-stack interaction with Tyr224 andstronger H-B with Lys254 than compound 11.

Those persons skilled in the art will appreciate that in order to studythe influence of the 3-D conformation of cyclopenta[d]pyrimidine agentson the tubulin antiproliferation activity, we devised three methoxynaphthyl substituted cyclopenta[d]pyrimidine compounds with maximumstructural similarity. Among them, theN,2-dimethyl-N-(6′-methoxynaphthyl-1′-amino)-cyclopenta[d]pyrimidin-4-amine(11) shows the rotation hindrance of naphthyl ring withcyclopenta[d]pyrimidine ring, and it adopts orthogonal angle of 101.7°.TheN,2-dimethyl-N-(5′-methoxynaphthyl-2′-amino)-cyclopenta[d]pyrimidin-4-amine(13) shows the free rotation capability of naphthyl ring withcyclopenta[d]pyrimidine ring, and it adopts orthogonal angle of 105.2°.The orthogonal and rotational ability difference of 11 and 13 leads tothe difference of binding affinity in colchicine binding site ontubulin, and completely difference antitumor activity (11 is inactivewhile 13 is extremely potent). We have established a method by combining2-D ¹HNMR (NOESY) and computer simulation to explore 3-D conformationinfluence on binding affinity.

It will be appreciated by those skilled in the art that changes could bemade to the embodiments described above without departing from the broadinventive concept thereof. It is understood, therefore, that thisinvention is not limited to the particular embodiments disclosed, but itis intended to cover modifications that are within the spirit and scopeof the invention, as defined by the appended claims.

What is claimed is:
 1. A compound of Formula III:

and 5,6-saturated and unsaturated with respect to the five memberedring; R₁ and R₂ may be the same or different and each is one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen; wherein (i) and(j) may optionally be attached to Y via a CH₂ bridge; wherein R₁ and R₂are not each hydrogen (H) when Y is N; R₃ is one of (a) a hydrogen (H),(b) a halogen, (c) an alkyl having from one to ten carbon atoms andhaving a straight or a branched configuration, and wherein the alkyl ispartially or completely saturated; (d) an NH₂, (e) an NHR₇, (f) anNR₇R₈, (g) an OH, (h) an OR, (i) an SH, and (j) an SR, and wherein R isone of R₁, and wherein R₇ and R₈ may be the same or different and is oneof R₁; R₅ is one of R₁, except when R₆ is a hydrogen (H), heteroaryl, orphenyl, wherein the phenyl and heteroaryl are optionally substitutedwith 1-2 moieties independently selected from the group consisting ofhalogen, (C₁-C₄)alkyl, (C₁-C₄)fluoroalkyl, (C₁-C₄)alkoxy,(C₁-C₄)alkylamine, and (C₁-C₄)dialkylamine, then R₅ is not (i) ahydrogen (H), or (ii) a phenyl group substituted at any position with asubstituent selected from the group consisting of (a) a hydrogen, (b) ahalogen, (c) CN, (d) OH, (e) NH₂, (f) (C₁-C₄)alkyl, (g)(C₃-C₆)cycloalkyl, (h) (C₁-C₄)fluoroalkyl, (i) (C₁-C₄)alkoxy, (j)(C₁-C₄)alkylamine, (k) (C₁-C₄)dialkylamine, (l) C(O)OH, (m) C(O)—NH₂,(n) C(O)—(C₁-C₄)alkyl, (O)C(O)—(C₁-C₄)fluoroalkyl, (p)C(O)—(C₁-C₄)alkylamine, and (q) C(O)—(C₁-C₄)alkoxy; R₆ is one of R₁; Xis a carbon (C) or a CR₄; and Y is one of (a) a nitrogen (N), (b) anoxygen (O), (c) a sulfur (S), and (d) a CR₆, wherein R₆ is one of R₁ andR₃ except when Y is CR₆ then said R₆ is not H when R₁ and R₂ are each H,and wherein when Y is O or S then R₂ is absent or is zero.
 2. Thecompound of claim 1 comprising pharmaceutically acceptable salts,hydrates, and solvates thereof.
 3. The compound of claim 1 wherein R₁ iseither a hydrogen or said alkyl having from one to ten carbon atoms andR₂ is a substituted naphthyl, or wherein R₁ is a substituted naphthyland R₂ is either a hydrogen or said alkyl having from one to ten carbonatoms.
 4. The compound of claim 3 wherein said substituted naphthyl is amethoxynaphthyl.
 5. The compound of claim 4 wherein said R₁ is saidmethoxynaphthyl and said R₂ is CH₃ and wherein said 5,6 bond is a singlebond.
 6. A method of treating a patient diagnosed with cancer comprisingadministering to the patient a therapeutically effective amount of acompound of Formula III:

and 5,6-saturated and unsaturated with respect to the five memberedring; R₁ and R₂ may be the same or different and each is one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen; wherein (i) and(j) may optionally be attached to Y via a CH₂ bridge; wherein R₁ and R₂are not both hydrogen (H) when Y is N; R₃ is one of (a) a hydrogen (H),(b) a halogen, (c) an alkyl having from one to ten carbon atoms andhaving a straight or a branched configuration, and wherein the alkyl ispartially or completely saturated; (d) an NH₂, (e) an NHR₇, (f) anNR₇R₈, (g) an OH, (h) an OR, (i) an SH, and (j) an SR, and wherein R isone of R₁, and wherein R₇ and R₈ may be the same or different and is oneof R₁; R₅ is one of R₁, except when R₆ is a hydrogen (H), heteroaryl, orphenyl, wherein the phenyl and heteroaryl are optionally substitutedwith 1-2 moieties independently selected from the group consisting ofhalogen, (C₁-C₄)alkyl, (C₁-C₄)fluoroalkyl, (C₁-C₄)alkoxy,(C₁-C₄)alkylamine, and (C₁-C₄)dialkylamine, then R₅ is not (i) ahydrogen (H), or (ii) a phenyl group substituted at any position with asubstituent selected from the group consisting of (a) a hydrogen, (b) ahalogen, (c) CN, (d) OH, (e) NH₂, (f) (C₁-C₄)alkyl, (g)(C₃-C₆)cycloalkyl, (h) (C₁-C₄)fluoroalkyl, (i) (C₁-C₄)alkoxy, (j)(C₁-C₄)alkylamine, (k) (C₁-C₄)dialkylamine, (l) C(O)OH, (m) C(O)—NH₂,(n) C(O)—(C₁-C₄)alkyl, (O)C(O)—(C₁-C₄)fluoroalkyl, (p)C(O)—(C₁-C₄)alkylamine, and (q) C(O)—(C₁-C₄)alkoxy; R₆ is one of R₁; Xis a carbon (C) or a CR₄; and Y is one of (a) a nitrogen (N), (b) anoxygen (O), (c) a sulfur (S), and (d) a CR₆, wherein R₆ is one of R₁ andR₃ except when Y is CR₆ then said R₆ is not H when R₁ and R₂ are each H,and wherein when Y is O or S then R₂ is absent or is zero, for achievinga therapeutic response.
 7. The method of claim 6 including administeringto the patient a therapeutically effective amount of a pharmaceuticalsalt, solvate, or hydrate of said compound of Formula III.
 8. A methodfor inhibiting the mitosis of one or more cancerous cells comprisingsubjecting one or more live cancerous cells to a mitotic inhibitoryacceptable amount of a compound of Formula III, or a pharmaceuticalsalt, solvate, or hydrate of the compound of Formula III:

and 5,6-saturated and unsaturated with respect to the five memberedring; R₁ and R₂ may be the same or different and each is one of (a) ahydrogen (H), (b) an alkyl having from one to ten carbon atoms andhaving a straight or branched configuration, and wherein the alkyl ispartially or completely saturated, or a substituted alkyl having fromone to ten carbon atoms, (c) a cycloalkyl having from three to tencarbon atoms, or a substituted cycloalkyl having from three to tencarbon atoms, (d) an alkylcycloalkyl, or a substituted alkylcycloalkyl,(e) an aryl, or a substituted aryl, (f) an alkylaryl, or a substitutedalkylaryl (g) a heteroaryl, or a substituted heteroaryl, (h) analkylheteroaryl, or a substituted alkylheteroaryl, (i) an aromatic, or asubstituted aromatic, and (j) a heteroaromatic, or a substitutedheteroaromatic, and wherein each substituent of any said substitutedgroup is the same or different and is selected from the group consistingof a straight or branched alkyl, alkenyl, or alkynyl, a cyclic oralicyclic group having from one to six carbon atoms, a heterocyclicgroup having from one to six carbon atoms, an alkoxy group, an aryloxygroup, an alkyloxyaryloxy group, an aryl group, an amine, a halogen, aphenol, a naphthalene, a piperidine, a pyrrole, a ketone, a methylalkylketone, and a trifluoromethyl ketone, and wherein each of saidsubstituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen; wherein (i) and(j) may optionally be attached to Y via a CH₂ bridge; wherein R₁ and R₂are not both hydrogen (H) when Y is N; R₃ is one of (a) a hydrogen (H),(b) a halogen, (c) an alkyl having from one to ten carbon atoms andhaving a straight or a branched configuration, and wherein the alkyl ispartially or completely saturated; (d) an NH₂, (e) an NHR₇, (f) anNR₇R₈, (g) an OH, (h) an OR, (i) an SH, and (j) an SR, and wherein R isone of R₁, and wherein R₇ and R₈ may be the same or different and is oneof R₁; R₅ is one of R₁, except when R₆ is a hydrogen (H), heteroaryl, orphenyl, wherein the phenyl and heteroaryl are optionally substitutedwith 1-2 moieties independently selected from the group consisting ofhalogen, (C₁-C₄)alkyl, (C₁-C₄)fluoroalkyl, (C₁-C₄)alkoxy,(C₁-C₄)alkylamine, and (C₁-C₄)dialkylamine, then R₅ is not (i) ahydrogen (H), or (ii) a phenyl group substituted at any position with asubstituent selected from the group consisting of (a) a hydrogen, (b) ahalogen, (c) CN, (d) OH, (e) NH₂, (f) (C₁-C₄)alkyl, (g)(C₃-C₆)cycloalkyl, (h) (C₁-C₄)fluoroalkyl, (i) (C₁-C₄)alkoxy, (j)(C₁-C₄)alkylamine, (k) (C₁-C₄)dialkylamine, (l) C(O)OH, (m) C(O)—NH₂,(n) C(O)—(C₁-C₄)alkyl, (O)C(O)—(C₁-C₄)fluoroalkyl, (p)C(O)—(C₁-C₄)alkylamine, and (q) C(O)—(C₁-C₄)alkoxy; R₆ is one of R₁; Xis a carbon (C) or a CR₄; and Y is one of (a) a nitrogen (N), (b) anoxygen (O), (c) a sulfur (S), and (d) a CR₆, wherein R₆ is one of R₁ andR₃ except when Y is CR₆ then said R₆ is not H when R₁ and R₂ are each H,and wherein when Y is O or S then R₂ is absent or is zero, for achievinginhibition of mitosis of said cancerous cell(s).
 9. A compoundcomprising Formula IV:

and 6,7-saturated and unsaturated R₁ and R₂ may be the same or differentand comprises one of (a) a hydrogen (H), (b) an alkyl having from one toten carbon atoms and having a straight or branched configuration, andwherein the alkyl is partially or completely saturated, or a substitutedalkyl having from one to ten carbon atoms, (c) a cycloalkyl having fromthree to ten carbon atoms, or a substituted cycloalkyl having from threeto ten carbon atoms, (d) an alkylcycloalkyl, or a substitutedalkylcycloalkyl, (e) an aryl, or a substituted aryl, (f) an alkylaryl,or a substituted alkylaryl (g) a heteroaryl, or a substitutedheteroaryl, (h) an alkylheteroaryl, or a substituted alkylheteroaryl,(i) an aromatic, or a substituted aromatic, and (j) a heteroaromatic, ora substituted heteroaromatic, and wherein each substituent of any saidsubstituted group is the same or different and is selected from thegroup consisting of a straight or branched alkyl, alkenyl, or alkynyl, acyclic or alicyclic group having from one to six carbon atoms, aheterocyclic group having from one to six carbon atoms, an alkoxy group,an aryloxy group, an alkyloxyaryloxy group, an aryl group, an amine, ahalogen, a phenol, a naphthalene, a piperidine, a pyrrole, a ketone, amethylalkyl ketone, and a trifluoromethyl ketone, and wherein each ofsaid substituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen; wherein (i) and(j) may optionally be attached to Y via a CH₂ bridge; R₃ comprises oneof (a) a hydrogen (H), (b) a halogen, (c) an alkyl having from one toten carbon atoms and having a straight or a branched configuration, andwherein the alkyl is partially or completely saturated; (d) an NH₂, (e)an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR, (i) an SH, and (j) an SR,and wherein R comprises one of R₁, and wherein R₇ and R₈ may be the sameor different and comprise one of R₁; R₄ comprises one of (a) R₁, (b) ahalogen, (c) a mono-, di-, tri- or tetra-substituted alkyl, and (d) analkyloxy, and wherein R₁ is H or a lower alkyl and R₂ is H or a loweralkyl then R₄ comprises one of (a) a NR₆R₇, (b) a SR₆, (c) a OR₆, and(d) a CHR₆R₇, wherein R₆ and R₇ may be the same or different andcomprise one of R₁ and R₂; R₅ comprises one of R₁; R₆ comprises one ofR₁; X is a carbon (C) or a CR₄; and Y comprises one of (a) a nitrogen(N), (b) an oxygen (O), (c) a sulfur (S), and (d) a CR₆, wherein R₆comprises one of R₁ and R₃.
 10. The compound of claim 9 comprisingpharmaceutically acceptable salts, solvates, and hydrates thereof. 11.The compound of claim 9 wherein R₁ is either a hydrogen or said alkylhaving from one to ten carbon atoms and R₂ is a substituted naphthyl, orwherein R₁ is a substituted naphthyl and R₂ is either a hydrogen or saidalkyl having from one to ten carbon atoms.
 12. The compound of claim 11wherein said substituted naphthyl is a methoxynaphthyl.
 13. A method oftreating a patient diagnosed with cancer comprising administering to thepatient a therapeutically effective amount of a compound of Formula IV:

and 6,7-saturated and unsaturated R₁ and R₂ may be the same or differentand comprises one of (a) a hydrogen (H), (b) an alkyl having from one toten carbon atoms and having a straight or branched configuration, andwherein the alkyl is partially or completely saturated, or a substitutedalkyl having from one to ten carbon atoms, (c) a cycloalkyl having fromthree to ten carbon atoms, or a substituted cycloalkyl having from threeto ten carbon atoms, (d) an alkylcycloalkyl, or a substitutedalkylcycloalkyl, (e) an aryl, or a substituted aryl, (f) an alkylaryl,or a substituted alkylaryl (g) a heteroaryl, or a substitutedheteroaryl, (h) an alkylheteroaryl, or a substituted alkylheteroaryl,(i) an aromatic, or a substituted aromatic, and (j) a heteroaromatic, ora substituted heteroaromatic, and wherein each substituent of any saidsubstituted group is the same or different and is selected from thegroup consisting of a straight or branched alkyl, alkenyl, or alkynyl, acyclic or alicyclic group having from one to six carbon atoms, aheterocyclic group having from one to six carbon atoms, an alkoxy group,an aryloxy group, an alkyloxyaryloxy group, an aryl group, an amine, ahalogen, a phenol, a naphthalene, a piperidine, a pyrrole, a ketone, amethylalkyl ketone, and a trifluoromethyl ketone, and wherein each ofsaid substituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen; wherein (i) and(j) may optionally be attached to Y via a CH₂ bridge; R₃ comprises oneof (a) a hydrogen (H), (b) a halogen, (c) an alkyl having from one toten carbon atoms and having a straight or a branched configuration, andwherein the alkyl is partially or completely saturated; (d) an NH₂, (e)an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR, (i) an SH, and (j) an SR,and wherein R comprises one of R₁, and wherein R₇ and R₈ may be the sameor different and comprise one of R₁; R₄ comprises one of (a) R₁, (b) ahalogen, (c) a mono-, di-, tri- or tetra-substituted alkyl, and (d) analkyloxy, and wherein R₁ is H or a lower alkyl and R₂ is H or a loweralkyl then R₄ comprises one of (a) a NR₆R₇, (b) a SR₆, (c) a OR₆, and(d) a CHR₆R₇, wherein R₆ and R₇ may be the same or different andcomprise one of R₁ and R₂; R₅ comprises one of Ri; R₆ comprises one ofR₁; X is a carbon (C) or a CR₄; and Y comprises one of (a) a nitrogen(N), (b) an oxygen (O), (c) a sulfur (S), and (d) a CR₆, wherein R₆comprises one of R₁ and R₃, and wherein when Y comprises O or S then R₂is absent or is zero, for achieving a therapeutic response.
 14. Themethod of claim 13 including administering to the patient atherapeutically effective amount of a pharmaceutically acceptable salt,solvate, or hydrate of said compound of Formula IV.
 15. A method forinhibiting the mitosis of one or more cancerous cells comprisingsubjecting one or more live cancerous cells to a mitotic inhibitoryamount of a compound of Formula IV, or a pharmaceutical salt, solvate,or hydrate of the compound of Formula IV:

and 6,7-saturated and unsaturated R₁ and R₂ may be the same or differentand comprises one of (a) a hydrogen (H), (b) an alkyl having from one toten carbon atoms and having a straight or branched configuration, andwherein the alkyl is partially or completely saturated, or a substitutedalkyl having from one to ten carbon atoms, (c) a cycloalkyl having fromthree to ten carbon atoms, or a substituted cycloalkyl having from threeto ten carbon atoms, (d) an alkylcycloalkyl, or a substitutedalkylcycloalkyl, (e) an aryl, or a substituted aryl, (f) an alkylaryl,or a substituted alkylaryl (g) a heteroaryl, or a substitutedheteroaryl, (h) an alkylheteroaryl, or a substituted alkylheteroaryl,(i) an aromatic, or a substituted aromatic, and (j) a heteroaromatic, ora substituted heteroaromatic, and wherein each substituent of any saidsubstituted group is the same or different and is selected from thegroup consisting of a straight or branched alkyl, alkenyl, or alkynyl, acyclic or alicyclic group having from one to six carbon atoms, aheterocyclic group having from one to six carbon atoms, an alkoxy group,an aryloxy group, an alkyloxyaryloxy group, an aryl group, an amine, ahalogen, a phenol, a naphthalene, a piperidine, a pyrrole, a ketone, amethylalkyl ketone, and a trifluoromethyl ketone, and wherein each ofsaid substituents may itself be substituted, and wherein any of saidsubstituents may be optionally attached by a CH₂ bridge, and wherein thesubstituent may be optionally partially or completely saturated orunsaturated when it is not represented by said halogen; wherein (i) and(j) may optionally be attached to Y via a CH₂ bridge; R₃ comprises oneof (a) a hydrogen (H), (b) a halogen, (c) an alkyl having from one toten carbon atoms and having a straight or a branched configuration, andwherein the alkyl is partially or completely saturated; (d) an NH₂, (e)an NHR₇, (f) an NR₇R₈, (g) an OH, (h) an OR, (i) an SH, and (j) an SR,and wherein R comprises one of R₁, and wherein R₇ and R₈ may be the sameor different and comprise one of R₁; R₄ comprises one of (a) R₁, (b) ahalogen, (c) a mono-, di-, tri- or tetra-substituted alkyl, and (d) analkyloxy, and wherein R₁ is H or a lower alkyl and R₂ is H or a loweralkyl then R₄ comprises one of (a) a NR₆R₇, (b) a SR₆, (c) a OR₆, and(d) a CHR₆R₇, wherein R₆ and R₇ may be the same or different andcomprise one of R₁ and R₂; R₅ comprises one of R₁; R₆ comprises one ofR₁; X is a carbon (C) or a CR₄; and Y comprises one of (a) a nitrogen(N), (b) an oxygen (O), (c) a sulfur (S), and (d) a CR₆, wherein R₆comprises one of R₁ and R₃, and wherein when Y comprises O or S then R₂is absent or is zero, for achieving inhibition of mitosis of saidcancerous cell(s).
 16. The method of claim 15 including administering tothe patient a therapeutically effective amount of a pharmaceuticallyacceptable salt, solvate, or hydrate of said compound of Formula IV.